FISHERIES RESEARCH BOARD OF CANADA
Translation Series No. 572
Digestive enzymes of freshly-caught codfish
by G. Siebdrt, R.V. Malortie, and R. Beyer
Original title: Verdauungsenzyme frischgefangener Dorsche
Prom: Arch. Fischereiwiss XITI: 21-34, 1962
Translated by Translation Bureau(EC)
Foreign Languages Division
Department of the Secretary of State of Canada
Fisheries Research Board of Canada
1964
27 pages typescript
7 ./(1A9 WO.
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DePARTMENT OF THE SECRETARY OF STÀTE
dUREAU FOR TRANSLATIONS
,
FOREIGN LANGUAGES
DIVISION
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INTO •-•
TRANSLATED FROM •- TRADUCTION DE
ENGLISH'
GERMAN
SUBJECT •-• SUJET
Fisheries Research
AUTHOR •-• AUTEUR
Siebert,,G., R. v, Malortie and.R. Bayer
TITLE IN ENGLISH •• TITRE ANGLAIS
DIGESTIVE ENZYMES OF FRESHLY-CAUGHT CODFISH
TITLE IN FOREIGN LANGUAGE - TITRE EN LANGUE eTRANGÉRE
Verdauungsenzyme frischgefangener Dorsche
REFERENCE •-• ReFÉRENCE (NAME OF BOOK OR PUBLICATION •• NOM DU LIVRE OU PUBLICATION)
PUBLISHER ••• ÉDITEUR
Arch. Fischereiwiss., XIII, 1/2, 21-34, Berlin,
Oktober 1962
Aus dem Physiologisch-chemischen Institut der
Johannes Gutenberg-Universitat, Mainz - (Dir.: Prof.Dr.Dr.
K. Lang)
CITY ••• VILLE
Berlin
DATE
received 12/12/1960
PAGES
21 - 34
REQUEST RECEIVED FROM
REQUIS PAR
of Fisheries
DEPARTMENT
MINISTÉRE
YOUR NUMBER
VOTRE DOSSIER N°
DATE RECEIVED
REÇU LE
41
el,
`-
105..20 0■ 10•
Mr. Paul Larose
7 69-18-14
sent 31 August, 1964
OUR NUMBER
NOTRE DOSSIER N°
6657.
Elisabeth Czeija
TRANSLATOR
TRADUCTEUR
DATE COMPLETED
REMPLIE LE
18/3/ 19 65
,
,Vo 7Z
• "
-
.
Arch. Fischereiwiss.
XIII
1/2
21-34
Berlin, Oktober 1962
(Fisheries Research Archives, Vol. XIII, No. 1/2, pp. 21-34,
Berlin, October, 1962).
"Aus dem Physiologisch-chemischen Institut der JohannesGutenberg-UniversitUt, Mainz"(From the PhysiologicalChemical Institute of the Johannes-Gutenberg University,
Mainz)
"(Dir.: Prof. Dr. Dr. K. Lang)"
"VERDAUUNGSENZYME FRISCHGEFANGENER DORSCHE"
( DIGESTIVE ENZYMES OF FRESHLY-CAUGHT CODFISM )
("Untersuchungen zur Verhiltung des Fischverderbs") 1 )
(Investigations concerning prevention of fish-decay)
by
G. Siebert, R. v. Malortie and R. Beyer
with 5 illustrations and 3 tables
Received on 12 December, 1960
A. Introduction
All deliberations regarding the preserva .„ion of
the quality of fish, i.e. for the purpose of avoiding decay, have to take into consideration the following two
factors:
Fermentative decomposition and bacterial attack
on fish muscle.
These two factors may also be differenti-
ated as an endogenous (enzymes) and an exogenous (micro
organisms) constituent (1, 10).
Moreover, the fermenta-
tive decomposition is creating a favourable environment
1)
To Prof. Dr. J. Kühnau with gratitude and reverence,
on the occasion of his 60th birthday.
.
,
1
2
ORO
for the subsequent penetration and growth of the bacteria.
(10).
Within the framework of the research on the enzymes of the fish muscle (2;3) we'asked ourselves the
question as to the extent to which digestive enzymes of
fish may contribute tocbcay within the period
of time
between the heaving of the haul on deck and the processing of the fishes (gutting, etc.). During this
period of time the fishes are stored on board, partly
under considerable pressure, they are not cooled with
ice, and in addition, they may possibly be damaged mechanically by the movements of the ship.
If, under these conditions which are briefly
called "storage aboard" in this report, enzymes of the
digestive tract are causatively contributing to the decay, and thereby to the deterioration of the quality,
it is to be expected that they
tract.
cavity.
cbme from the digestive
Hence, they should first be found in the body
If this is the case to aryappreciable degree,
they might also be able to enter into the surrounding
musculature from the body cavity.
On the basis of these deliberations, we examined
fishes which had been stored aboard,
and others which
had not been stored aboard, respectively.
The fisheries
management stated that the period of storage aboard
does not exceed two hours,œ a rule.
to be described here,
For the experiments
the period of storage aboard was
intentionally extended Up to eight hours so as to gain
a clearer insight into the processes occurring during
storage aboard, in the light of extreme conditions.
2:22
•-• •
3
As preliminary experiments with freshwater
fishes had shown that this question is obviously significant, extensive experiments were made on codfish
within a large-scale investigation at sea.
Codfish
("Gadus callarias (= morrhua L.1 was selected because it
is available in sufficient number, and as it is a food
product of considerable economic importance. The body
cavity was rinsed out and thus the enzymes possibly contained therein were obtained as research material.
Muscle
areas near the body cavity served to detect a possible
entry of digestive enzymes from the body cavity into their
vicinity.
Dorsal musculature was used to check on changes
caused by storage which are not due to the influence of
digestive enzymes.
B.
Methodology
Samples obtained: Due to the obligingness of the
authorities concerned, the samples were obtained during
a journey of the research ship "Anton Dohrn" (under the
direction of Dr. K. Schubert). Out of each haul (30 min.
dragging time) two codfish. were taken immediately, and
treated as described below. Another six animals were
placed in a basket on board in such a way that theyAriere
covered by a layer of other fishes, about 30 cm high,
the pressure exterted on the animals in this way corresponds to the storage of animals for research purposes
in the lower third of a deck pond ('*).
After two,
four and eight hours, two animals were taken out after
each interval, and were also treated as described below.
Translator's note: The German word given here is:
"Hock", apparently a fish-container.
mi,Yee,(.1
-4-.
In this procedure, the fishes were placed on the
working table between two wedges, with the abdomen upward.
The abdominal flesh was raised with forceps, without raising
the intestines at the same time, and was opened
above
the anus by a crosscut r-2cm long, and by a longitudinal
section, 3 cm long, toward the head. According to the size
of the fish (not less than 35 cm), 10 or 20 ml of a 9%
sodium chloride solution was carefully injected into this
opening with a hypodermic syringe. Both side3of the opening
uere/seized with the forcepses, and the fish was briefly
shaken. Then this rinsing fluid was taken out again with the
hypodermic syringe (blunt, without cannula), the quantity
of the liquid noted, filled into the sample bottle, and
frozen immediately at --25 ° C.
It was impossible to de-
termine with this method, as to whether or not any damage
had occurred inside the abdominal cavity due to the storage
However, adulteration of the rinsing fluid
under pressure.
by the intestinal contents is out of the question. A reddish
shimmer of some rinsing fluids can probably be traced to
impurities due to blood which entered into the abdominal
cavity after a vessel had burst. Then the 3 cm-long longitudinal section was further extended with a knife up to the
p.23
pectoral fins.
Then two cross sections were made on the
left side, running from the upper edge of the abdominal
cavity to the ends of the longitudinal section . The muscle
area which had been loosened in this way was then flapped
down and detached by a further longitudinal section/at the
abdominal cavity.
The peritoneum was then carefully re-
moved and a strip, r-,d 2 cm wide,r,d5 cm long and,—.i5 mm
thick, was cut out from the muscle. This specimen was also
frozen immediately in the deep freeze chest, right after
•
5
The samples from the dorsal
having been obtained.
musculature were also disengaged from the muscles by a
trapezoidal incision in the dorsal skin and by detaching
the skin neatly. Then a cube, with edges approximately
2 cm long, was cut out as a specimen from the centre of
the muscles
set free, and was frozen immediately.
The places where the fishes were caught are
located in the southern half of the North Sea, especially
between 7 0 eastern and 1 ° western lonEitude, 55 0 and 56 0
north latitude. The period during which the fish were
caught extended from 14 September to 12 October, 1960.
More detailed information is found in the minutes kept
by the director of the expedition.
In general, all the animals examined had just
eaten to judge from the degree to which the digestive
tract was filled.
No data are available with regard to
the stages of maturity.
The frozen test samples, a total of more than
300, were transported to Mainz, under deep cooling, and
were stored in frozen condition until the exanination.
Enzymes were determined in the following way:
Trypsin (4):
Either 0.5 ml rinsing fluid,or
0.5 ml of a 20% muscle homogenate in 1% KC1
5 X 10 -3
M Versen*, serve as enzyme source.
The incubation mixture contains the following:
0.5 ml enzyme
0.5 ml 0.1Mphosphate pH 7.6
*
Translator's note:
7m,1`.77
Versen might be a trade-name?
1.25 ml 1% casein (heat-denaturized)in the above
phosphate buffer.
The test temperature is 37° C.
The period of incubation is 30 and 60 min., which is then
interrupted with 2.75 ml 5% TCE.
After filtration 2 ml
are used for determination with the phenol reagens,
reading at 650 m/u.
The calibration curve is prepared with tyrosine.
Cathepsin (4).
The enzyme-source is the same as
above.
The incubation mixture contains the following:
0.5 ml enzyme,
0.5 ml 0.1 M acetate p H 4.3,
1.25 ml 3.3% Nb-solution (denaturized with alkali and urea)
PH
The temperature during the experiment is 37 0 C.
The period of incubation is 10 and 20 minutes, interrupted
and analyzed as above.
In case the activities are too high, the quantity of the
enzyme as well as the period of incubation are decreased.
Amylase (5). The source of the enzymais the same
as above.
The following is contained in the incubation mixture:
0.5 ml enzyme,
0.5 ml 0.02 M Na-glycerophosphate buffer pH 6.9,
1.0 ml 1% starch solution in the above buffer ("Amylum
solubile Merck").
The temperature during the experiment is 25 o C.
The period of incubation is 10 and 20 minutes, which is
then interrupted with 3.5-dinitro-salicylic acid in
p.2/A
NaOH (2 ml); after heating to 100 0 C during 5 minutes,
ad 20 ml is diluted with H 2 0 and read at 540 m/u.
calibration curve is made with maltose.
C.
The
Preliminary Experiments
Dr. H. Mann, federal fisheries research institute
in Hamburg, kindly provided the samples for the preliminary
experiments. Two freshly-caught breams
or trout, at a
time, were treated in the way described under Methodology;
the storage conditions on board can naturally not be
imitated'completely (pressure, temperature).
tained are summarized in Tables I and 2.
The data ob-
They show that
even only with a short period of storage trypsin occurs in
the body cavity
/and there would appear to be no doubt that it originates
in the digestive tract. Due to the limited number of
animals, it is impossible to draw statistical conclusions
from these experiments.
But the preliminary tests do not
exclude the possibility that trypsin also enters into the
abdominal musculature during storage.
Two supplementary findings are worth being mentioned
briefly: from the beginning a considerable cathepsin activity is found (in a physiological manner?) in the body
sic
cavity, in the case of breams. . In the case of both animals
certain increases in cathepsin activity in the musculature
occur during storage.
Table 1
Enzyme-changes during storage of bream's
(preliminary ex-
periments). (All values as ,y tyrosine/hours/g fresh weight
of muscle or entire body cavity respectively)
?7,77.7.f7 r7eC.
-E
oh
Duration of storage
2.5
h
5.75
h
8.75
h
24
h
Body Cavity
0
20
50
lo
60
94
180
180
140
160
0
0
160
10
10
20
25
49
36
18
Trypsin
0
. 0
10
0
10
Cathepsin
7
11
27
18
25
Trypsin
Cathepsin
Abdominal Musculature
Trypsin
Cathepsin
Dorsal Musculature
Table 2
Enzyme-changes during storage of trout, (preliminary experiments). (All values as ry tyrosine/hours/g
fresh
weight of the muscle or the entire body cavity respectively)
Duration of storage
0h
375 h
8.5 h
22h
o
40
540
96
Body Cavity
. Trypsin
.
Cathepsin
not tested
Abdominal Musculature
Trypsin
o
Cathepsin
o
•o
96
16
27
23
9
o
o
o
9
18
13
14
18
Dorsal Musculature
Trypsin
Cathepsin
D. Resalts
I. Trypsin in the body cavity
The body cavity of codfish contains, even without
p.25
storage on board, measurable amounts of trypsin-like
proteolytic activity.
The distribution of the individual
values is illustrated in Illustration la. In calculating
the data the rinsing-fluid-volume concerned had been taken
into consideration; the data, with a mean value of
280
170y tyrosin being liberated per hour, refer in each
case to the total proteolytic activity obtainable from the
Illustration 1 b shows
body cavity of each animal tested.
the distribution of the measuring values obtained for
trypsin after a 2-hour period of storage on board. One of
the values is already exceptionally high; the mean value
is thereby shifted to 830ey tyrosin/hours, which corresponds
to an increase to 300%.
Three measuring values lie outside
the scattering range of the three-fold medium error of the
The other 22 values
mean value of the 0-hours-animals.
do not differ significantly from the values measured without
storage aboard.
[12
to
e
t
ior,
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Trypsinaktivitidm•, , TyrosinStumle.LoibesWile
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CO
1
/11. lb
Illustrations la and lb
Trypsin activity in ry tyrosin/hour/body cavity
After a period of four hours of storage on board (Ill. 1c)
- 10-
The medium value
the picture is already quite different.
of ail measuring data is 2100y tyrosin/hours, this amount
again being partly caused by a value which had risen excessively high. For evaluating this series of measurements, the data obtained were divided into those which
still fall into the scattering range of the three-fold
mean error of the mean value of the animals without
storage on board (i.e. concerning the animais which have
gone through a 4-hour period of storage on board without
any noticeable damage), and those values lying above this
scattering range (10 animals out of a total of 25 animais).
a procedure would appear to be justified, as in view Such
of the question asked and the instructions for tha experiment, it can only be expected that the values either
rise upwards or that they remain at the initial level.
Out of the 10 values having risen above the norm, excluding
the one excessively high value, a mean value of 1880 If:
1320 7 tyrosin per hour and body cavity is obtained, which
is obviously significantly different from the initial
value.
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animals animals
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Ill. 2a
Ill. 2b
Ill. lc and Ill. id
activity inTrypsin 'Y
-
Amylase-activity in mg maltose/
hour/body cavity
tyrosin/hour/body cavity
After an 8-hour period of storage on board,fracti-
cally all the animals, with the exception of three, reached
values of trypsin-like activity lying above the initial
values (see Ill. id having a scale which is only half of
that in Ill. la to 1c).
If the one excessive value is
eliminated from the figures, the mean value obtained for
the remaining 25 animals amounts to 15 000
12 000 y
tyrosin per hour and body cavity,which differs significantly
from the initial value. The experiments with trypsin in
the body cavity show, therefore, that even without storage
on board a certain activity is measurable whidh rises to
300% after two hours, to 750% after four hours, and to
7900% after eight hours.
IL
Amylase in the body cavity.
Amylase-activity in the body cavity is unusually
small without storage on board (Ill. 2a).
of 26 animals, 20 show no activity.
Out of a total
The mean value is
calculated at 1.4 ± 0.13 mg maltose/hour, again with reference to the entire body cavity.
After storage on board for a period of two hours,
the amylase contents of the body cavity have risen already
considerably, to more than 500%,
but the scattering of
the values (see Ill. 2h) is so great that the mean value of
7.3
p.27
6.9 mg maltose/hour does not differ significantly
from the initial value without storage on board.
the 25 samples tested are without any activity.
,
•
' "
,
13 of
Amylase
.
•7-.My•:•,
- 12 increased still further after storage on board for a
The measuring values are entered
period of four hours.
in Ill. 2c. 15 of the 26 animals tested still show.negative values; 11 animals, however, have alreadyparUyvery
strong amylase activity. Although a sevenfold increase
of the mean value is noted, to 9.9 mg maltose/hour, the
difference in comparison with the initial value is not
IQ
p.
;
5
r- 4
3
;OS
11.1
■
I
70
de
Jr
sTettu ireJo aequinN
yet significant statistically.
o
7 ;5
2‘, 14
mu 2,
Illustration 2c
Ill. 2c
6
a)
0
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.;7
I
94
—
r
70 61
I
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9
.1b11. 2d
M.thnie
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I
-1e
•
"
P.3
1111 7.2d
"
A in} ia ,eakti. iti n mg
Ill. 2d
Amylase-activity in mg maltose/hour/body cavity
Even after storage on board over a period of
rî,g rY7M
- 13 eight hours, it was found that seven of the 24 animals
tested had no amylase activities in their body cavities.
The remaining animals, however, all showed considerably
increased activities (Ill. 2d) with a mean value of
18 j 14 mg maltose/hour per body cavity, which thus lies
high as does the initial value. The
thirteen times
differerehce is statistically significant.
Hence, amylase rises also considerably during
storage on board, but less so than trypsin.
III.
p.28
Cathepsin in the body cavity
Due to the findings in the case of fresh water
fish (see above), experiments were also made to determine
cathepsin-like proteolytic activity in the body cavity.
But in this case only animals without storage aboard were tested
In later stages, when the trypsin in the body cavity is
beginning to rise, there is a certain danger that the
measuring values for catheptic enzymes are superposed
by tryptic enzymes; it would only be possible to eliminate
this difficulty if a great amount were spent on the experiments which does not appear to be worth-while in this
case. The values obtained for cathepsin are depicted in
Illustration 3. The mean value from 25 animals is
760 4 350 ry tyrosine/hour per body cavity.
In contrast
with the trypsin tests (Ill , la), none of the animals
tested is found to be without any activity; the mean
value is almost three times as great as the mean value
for trypsine.
Thus the possibility of"cathepsir;gvalues
being simulated by "trypsin q-like enzymes is excluded.
Not excluded is the possibility of "pepsin"-like proteases
being responsible for the effect observed.
This would
mean that, considering the p H -value at which the experiment
)
1
,
- 14 was carried out, a peptic activity would have to be again
many times greater than the enzyme activity designated
here as "cathepsinn.
Experiments for the purpose of clari-
fying this question have to be deferred at the moment.
I- 4
I"
I I 1 III
1 90
22O) ,7 0
1.:?1)
120
AU, .
KatheF,qtinktiviii.t in
eco
e00
3 Ill . 3
e
—3›
-
H.
0
S
0'
M 0
1-5
y TyroainiSturid‘)/Leibe3hiinic
Illustration 3
Cathepsin-activity in 'y tyrosine/hour/body cavity
It follows from the experiments, however, that the
body cavity of cod does not only contain tryptic but also
cabheptic enzymes, and the latter to a degree of considerable
activity.
On the whole, therefore, it is to be expected
that, in accordance with the pH present in the body cavity,
that there is already substantial proteolytic capacity
existing, even without any damage havinE been inflicted
upon the animals.
The increase of trypsin and . amylase in the body
cavity during storage on board, as reported above, is
leading to the question as to whether or not there is any
relation between the two processes.
A priori it might be
assumed that the increase of one enzyme in the animal concerned should be connected with an increase of the other
enzyme in the . same animal. For this reason, the percentages
of the deviations of the enzyme activities from the mean
value of trypsin , or amylase respectively, were entered
in the coordination cross for each animal (Ill. 4), for
the case of storage on board for an eight-hour period
-1 5
when changes are most pronounced.
-
A calculation of the
data (6) results in a correlation coefficient of
0.49, which indicates only a rather loose
rr-'
connection, but in any case no counter evidence.
IV. Trypsin in the abdominal musculature
Even without any storage on board a slight trypsin
activity is present in the abdominal muscle. The average
amounts to 14 J 3.5/tyrosin/hour and g fresh muscle
tissue.
These findings agree with the unpublished ex-
periments on proteasesin fish muscle.(7)
p.29
( body cavity)
1-S00
+ire
o
I.- +200
-
AWL4
VcIAautu32
en....yme
in der
Le;hth..iii;)Mc 1-1,rh
adiAsiFiridiger
Huriilaviun•r.
o
0
,
i
.
,.--..
,
-:o0 -75 -50
d-e5
,ro
prozzuttuale .
Mtt iftb...ert !..iir
jodes eirmelni,
Tier
-2:;
Ae.yfesi,
(body cavity)
- - 50
Abweirliting‘"in
.
T—
aufg,trai.r.en Ibi
0
o
?
9
e' •
0
°
•
.
1.--co
I
.
Ill. 4 - Digestive enzymes in the body cavity after a period
of eight hours of storage on board, plotted on the diagram as
the percentage of the deviation from the mean value for each
single animal.
1
- 16 After storage on board for a period of two hours,
the trypsin activity in the abdominal muscle rose to
185% (26
4.9
y tyrosine/hour and g fresh tissue). The
difference is already significant as compared to the
initial value.
A further significant increase of trypsin activity
in the abdominal musculature to 120
11 y tyrosine/hour/
gfresh weight, i.e. 8.6 times as much, is found after
storage on board for a period of four hours.
The effect
is still considerably stronger after storage on board for
eight hours; in this case a mean value of 650 ± 107=y
tyrosine/hour/g fresh weight is reached, i.e. a 46-fold
increase is found.
For the purpose of clarifying the question as to
whether or not there is a connection between the trypsin
increase in the body cavity and that in the abdominal
musculature, the percentage of the deviation from the
mean value after storage on board for eight hours is
entered in Illustration 5 for each'animal tested. It
would appear that an increase of trypsin in the abdominal
musculature should only be found if the body cavity of
the animal concerned also contains more trypsin.
A check on the calculation of the data repre-
p.30
sented in Illustration 5 results in a correlation coefficient of r
0.55, a value which suggests a loose
connection, but excludes the possibility of a lack of
any connection.
Illustration 5
- Trypsin in the body cavity and
abdominal musculature after an 8-hour period of storage on
-
17
-
board, plotted on the diagram as the percentage of the
deviation from the mean value for each individual
animal.
Illustration 5
(see translation
beginning on preceding page - last
0 paragraph)
(body cavity)
Ptsre
U+409
1- +309
+290
o
o
-+790
o
a
1
• 1)0 -25
-4 90 7j5
Il
o
I
_26
— —50
+7100
--- i
+coo
-
1
-poo
,
rupsm
4-400
-, souch..kwaur
(abdominal muscu.turej
Abb. 5
Trypsin in
Leibesh6hle und
Bancliniuskulatur iiach
8stiindiger Bonibigerung, aufgetragen ids
prozentuale
Alweichung voila
Mittelwert far
jedes chuckle
Tier
e'i%
-
100
V. Amylase in the abdominal musculature.
Without storage on board the amylase activity
in the abdominal musculature amounts to 270 ± 25 -y
maltose/hour/g fresh tissile.
After storage on board
for eight hours, it increases by 30% to 350 j
maltose/hour/g fresh weight.
34
This difference is slight
but statistically significant. Because of the slight
difference of 30%, the samples of the abdominal muscles
were not tested after storage on board for periods of
two and four hours, as significant deviations from the
initial value cannot be expected to occur.
Cathepsin in the abdominal musculature was not
tested, as similarly to the case of the body cavity, a
-18superimposition of the measuring values by trypsin would
be possible to eliminate only if very considerable
amounts were spent on the experiments.
VI. Trypsin in the dorsal musculature
The trypsin activity found in the dorsal musculature is higher than that in the abdominal musculature.
2.8 'y tyrosine/hour/g fresh
The mean value is 35
tissue.
After storage on board for an eight-hour period,
this value increased by 30% to 47
--1--
3.8'y tyrosine/hour/
g fresh tissue. The difference is significant.
But the
n.t.2.1
increase in trypsin-activity due to storage on board is
approximatively 150 times larger in the abdominal muscle
than it is in the area of the dorsal muscle.
Amylase was not tested in the dorsal musculature.
Even in the abdominal musculature the increase is so
slight during the eight-hour period of storage on board,
that no new insights can be expected from testing
the
dorsal musculature.
VII. Cathepsin in the dorsal musculature
As is known (2,3) cathepsin activity is relatively
high in fish muscle; for the dorsal musculature of cod
it amounts to 425
197 tyrosine/hour/g fresh tissue,
and after storage on board for eight hours it increases
by 30% to 555 ± 87 'y tyrosine/hour/g fresh weight.
This
difference is statistically significant.
E. Discussion
For the purpose of giving a clearer summary of
the test results, another survey in figures is given in
Table 3 which follows.
The following conclusions may
- 19 be derived from them:
a)
A three- to five-fold increase of trypsin-
and amylase activity is reached in the body cavity after
Illustrations 1 and 2
storage on board for two hours.
indicate that individual animals show increased values
and contribute thereby to the increase of the mean
value of all animals tested.
h) In case of longer storage on board, the enzyme
activities in the body cavity increase enormously(Trypsin,
80-fo 1 d; amylase 13-fold).
A definite damage suffered by
the animals can already be noted here.
c) It is found that the longer the storage
period, the greater is the increase of the trypsin content,
finally up to 47 times the initial value. Amylase increases also, though only
.sligaty.
It may be assumed,
therefore, that under the experimental conditions chosen,
digestive enzymes penetrate as far as into the abdominal
musculature.
d) Trypsin increases somewhat in the dorsal
musculature, but only to the extent of 1/150th of the
amount of the increase in the abdominal musculature.
Hence it surely follows that the observations made in
the abdominal musculature can not be explained by changes
having occurred in the muscle tissue ïtSelf but r:ather by
the fact that enzymes enter from outside.
The experimental conditions chosen for this investigation are standardized to a certain extent in relation to the conditions prevailing, as a rule, on board
fisheries vessels, as only in this way clear results can
be expected.
The net-dragging period lasted only 30
- 20-
minutes, as compared to the normal period of about two
hours in the case of steam-trawlers. If the pressure
of a half-full or full net against the water during the
dragging period is taken into consideration (in these
experiments 4 knots at full . engine capacity:), it may
be assumed that the fishes are
subjected to considerable
mechanical stress already in the net (11).
is quite possible that finestlacerations
Hence, it
occur in the
digestive tract and facilitate the entry of enzymes.
It
must, therefore, be assumed that the fishes from the net
arrive on deck already damaged to a certain extent (11).
This may also be the reason why indiiridual animals which
were tested without prior storage on board, show greater
trypsin values in the body cavities (Ill. la).
Table 3
P.32
Changes occurring in enzymes when cod is stored on board
Period of storage
in hours
;
O
•
2
4
8
Body Cavity
Trypsin
ey tyrosine/hour/
280
body cavity
%edeviation_ • -:- •- , ._•
Amylase
ty Maltose/hour/ 1400 ± 130
body cavity
% of deviation
830*)
300
7300*)
520
2100**) . 22 000**)
750
7 900
9900*) 18000.1L14000*
710
1 290
Cathepsin
'y tyrosine/hour/ • 760 ± 350
body cavity
Abdominal musculature
Tryp sin
y tyrosine/hour/g
fresh weight
% of deviation
14 ± 3.5 26jL4.9**) 1201:11**) 650jE107**)
190
860
4 650
Amylase
y maltose/hour/g 270 4 25
fresh weight
% of deviation
3501 3 4**)
130
21 -
(continued:
Table 3)
Storage-period
in hours
0
2
8
4
Dorsal Musculature
Trypsin
,y tyrosine/hour/g
fresh weight
% of deviation
35
E
2.8
47 ± 3.6**)
130
Cathepsin
7 tyrosine/hour/g
425 ± 19
---
---
555 ± 87**)
fresh weight
% of deviation
*)
130
The difference as compared with the initial value is not
statistically significant.
**)
The difference from the initial value is statistically
significant.
The fishes were partly stored on board for a longer
period than the 2-hour period reported by the fisheries
management, before they were tested; and in this connection
the question remains open as to whether or not this storageperiod is extended also in practice, under special circumBut as Table 3 shows, even after a storage-period
stances.
of two hours the fishes show a measurable degree of damage.
The temperatures under which these experiments were carried
out probably correspond to those usually prevailing on board
Temperatur:e measurements in theàbdominal
cavities of the codfishes tested indicated 13 o C without
fishing vessels.
storage on board inaccordance with the water temperature.
It may be higher after :t orage on board. The amount of
pressure selected for the storage of fish in these experiments is not higher than that probably occurring in
practice.
Trypsiu, or trypsin-like protease are mentioned in
this report. An exact classification as trypsin would
- 22 presuppose the use of synthetic substrata and of a
trypsin-inhibitor instead of measuring proteolysis so as
to determine separately proteases other than trypsin. To
be correct, therefore, the definition "proteolytic activity
at pH 7.6" should have been chosen. It is, however,
irrelevant, even undesirable, for purposes of the present
experiment, to measure trypsin alone, as proteolytic activity in its totality is of interest.
The liberation of
tyrosine from casein is used for measuring the trypsinaction. It is justified, in general, to multiply the
quantity of tyrosine by the factor 10 to 20 so as to obtain
the amount of the decomposed protein.
If the factor 10 is p.33,
on board for two
0
hours, an average of 8 mg protein/hour at 37 0, i.e. about
1 - 2 mg protein/hour at+13 ° C can be decomposed in the
body cavity.
Trypsin splits protein into peptides, and in addition,
to a small extent, also into amino acids. Fish muscle is
extraordinarily rich in peptidases (8).
glycogen to maltose.
Amylase degrades
It is not known whether or not fish
muscle contains a maltase.
Hence, both enzymes tested
here are ideally suited to prepare a nutrient substrate
from amino acids and carbohydrates for bacteria. On the
basis of the results of the experiments, it is, therefore,
justified to draw the conclusion that in case of fish being
stored on board too long, the fish is transformed from foodstuff into a nutrient substrate for bacteria under the influence of its own digestive enzymes. The reader is referred to Lticke (10) with regard to the mutual interdependence
of germ diffusion and autolysis.
used,itfonhaevftrsoage
••••
•
.
- 23
Two additional effective qualities of trypsin
are to be noted in this context:
First, the firmness of
the muscular tissue of fish, which is in itself limited,
suffers further from the trypsin-action.
Thereby,
additional favourable conditions are created for the
bacteria (10) to enter from the skin, the intestine and
the gills. Hence, trypsin also serves to create an entrance door for bacteria.
It is known from human medicine that due to supply
with small quantities of trypsin e e.g. through the mucuous
membrane of the mouth, the proteolytic enzymes in the
in their preliminary stages
tissues/are transformed into their active forms(plasmino-
gen
plasmin) (9).
It isrilot known whether or not
the tissues of fish contain plasminogen, but it is to be
assumed until the absence of it is proven.
It might be
assumed that small quantities of trypsin, which enter into
the abdominal musculature cause great additional effects
(of an autolytic nature).
In these experiments no consideration was given
to the size of the animais (those used were from 35 cm in
length onward), the stage of maturity and the degree to
which the alimentary canal was filled up at the moment.
But it can be assumed that the more than 100 codfishes
tested here represent a useful average,with regard to
these properties, of all the cod caught in practice.
Under the terms of these experiments no enzymes
other than trypsin and amylase have been tested so far.
But the results obtained here would appear to lead to the
conjecture that, in gradual degrees, in principle,
identical results are also obtained with other digestive
enzymes.
,
r7eme,eM7Vre
- 2 4-
A supplementary finding which is striking is the
fact that obviously the body cavity normally contains
cathepsin.
It is not possible to decide on the basis of
the present experiments, if the cathepsin in the body
cavity is active or combined with an inhibiting substance.
For clarifying this question further, fishes are
to be examined which had not been exposed to possible
damage in dragging the net.
It would seem appropriate
to test other species of fish and to examine whether or
not other enzymes appear in the body cavity.
In practice, certain types of fish (herring, small
red perch) are not gutted when they arrive on land. As
smaller animals spoil more quickly than larger ones do
(1, 10), it would be advisable to apply the instructions
for the experiments used here to such fishes which had
not been gutted. During storage on board a not very
great, yet statistically secured increase of the
cathepsin activity is noted in the dorsal musculature,
i.e. far away from the body cavity.
One is tempted to
assume that there is some kind of "maturing process",
e.g. through the degradation of an inhibitor. Since
the cathepsin-activity is very considerable (about
1 - 2 mg protein/hour/g fresh muscle at 13 ° C), this
problem should perhaps also be studied more closely
under another experimental setup.
The authors wish to thank Messrs. Dr. Mann
2.1.321
and Dr. Schubert, "Bundesforschungsanstalt ffir Fischerei"
(federal fisheries research institute), Hamburg, Mr.
Preisler, "Deutsche Fischwerbung" (German fisheries
publicity), Bremerhaven, and the crew of "Anton Dohrn"
7.7
à
-
25
-
for their assistance in obtaining test samples. It is
noted with gratitude that the experiments were supported
financially by grants from the "Bundesministeriums fUr
ErnUhrung, Landwirtschaft und Forsten" (federal department of food, agriculture and forestry), and the
"Ernghrungswissenschaftlichen Beirabsder Deutschen
(advisory council for food research
Fischwirtschaft"
of the German fisheries trade).
F.
Summary
G. Summary
1.
Cod caught on séa were stored under con-
ditions usually existing on board. At certain times
the contents of the abdominal cavity was removed by
rinsing with saline.
Tissue samples were taken also
from the ventral and dorsal muscles.
2. During the storage on deck the digestive
enzymes in the abdominal cavity and in the surrounding
muscles show a considerable increase.
3.
Storage on deck, even for 2 hours only,
damages freshly caught cod to such a degree, that
they are favourable for bacteria growth, thus spoiling
far more easily.
4.
Similar conditions can be observed with
freshwater fish.
*
Translator's Note: The summary given under "G" in
English in the original text is a translation of the summary
under "F".
■1717,77,.,Ve",
•
.e"...77■1 ■ 1,-,
1,PyMeta»re -..,m
-
26
-
5. The abdominal cavity of cod contains, even
under normal conditions, cathepsin 4 When stored on deck,
the activity of the proteolytic enzymes in the dorsal
muscles increases.
The importance of these processes
is discussed.
6. The investigations have the purpose to encourage measures for maintaining the quality in a better
way. The investigations have clearly shown that there
is an opportunity to improve the storage conditions on
deck.
H. Bibliography
(1) Sander, G.: Abh. Fischerei u. Hiifswiss.
(Treatise
on fisheries and auxiliary research) 3, 613 (1950).
(2) Siebert, G.: Exper. 14, 65 (1958).
(3) Siebert, G.: Arch. Fischereiwiss. (Arch. fisheries
research). 8, Beiheft (additional booklet) 1,
43 (1957).
(4) Laskowski, M.: Meth. Enzymol. 2, 26.
(5) Stein, E. A. and E. A. Fischer: J. biol. Chem.
232, 867 (1958).
(6) Koller, S.: Graphische Tafeln zur Beurteilung
statistischer Zahlen (Diagrams for interpreting
statistical figures). p. 9f. Darmstadt, 1953.
M'7
(7)
Siebert, G. and R. v. Malortie:
Malortie unpublished.
(8)
Schmitt, A. and G. Siebert
Siebert: unpublished.
•
-
(9) Innerfield t J.:
27
-
Enzymes in Clinical Medicine. 000.
(10) Lücke, F. and E. Frercks: Vorratspflege u. Lebensmittelforsch.(Care of stored provisions and
food research). 3, 130 (1940).
(11) Llicke, F. and W. Schwartz: Arch. Mikrobiol.
(Arch. Microbiol.). 8, 207 (1937).
MIY17.
mM7r,MM.-