On the prevention of the black spot of shrimp. 1. Effect of some chemical reagents on the black spot of shrimp. 2. The isolation and purification of the enzyme related to the formation of black spot


Ito, Y.

Boletim do Instituto Oceanografico Sao Paulo, 16: 1-11

1967


The causes of the black discoloration of three species of shrimp commonly caught off the coast of the State of Sao Paulo, Brazil, are studied and measures for its prevention are advocated. Landings of shrimp in Santos in 1964 equaled 13% of the total landing, by weight, while economically it is the principal fishery. I — Blackening was effectively prevented by dipping the shrimp in cooled chemical solution for 30 minutes and then storing them at low temperatures. L-ascorbic acid gave the best results, delaying discoloration up to 9 days at 0° to —2°C and for a longer period at colder temperatures. The effect of Sodiumthiosulfate and EDTA at different temperatures is also discussed. II — Tyrosinase was isolated and assayed by measuring the formation of Dopα-chrome from DL-Dopa at pH 6.8 an 37°C. Tyrosinase was obtained from the shrimp liver and was purified by approximately 25 times its activity from the initial extract by absorption chromatography through Celite 545, as judged by the rate of dopachrome formation.

ON
THE
PREVENTION
OF
THE
BLACK
SPOT
OF
SHRIMP.
I.
EFFECT
OF
SOME
CHEMICAL
REAGENTS
ON
THE
BLACK
SPOT
OF
SHRIMP.
II.
THE
ISOLATION
AND
PURIFICATION
OF
THE
ENZYME
RELATED
TO
THE
FORMATION
OF
BLACK
SPOT.
(Received
in
29/4/1966)
YASUZO
ITo
**
SYNOPSIS
The
causes
of
the
black
discoloration
of
three
species
of
shrimp
commonly
caught
off
the
coast
of
the
State
of
Sao
Paulo,
Brazil,
are
studied
and
measures
for
its
prevention
are
advocated.
Landings
of
shrimp
in
Santos
in
1964
equaled
13%
of
the
total
landing,
by
weight,
while
economically
it
is
the
principal
fishery.
I
Blackening
was
effectively
prevented
by
dipping
the
shrimp
in
cooled
chemical
solution
for
30
minutes
and
then
storing
them
at
low
temperatures.
L
-ascorbic
acid
gave
the
best
results,
delaying
discoloration
up
to
9
days
at
to
—2°C
and
for
a
longer
period
at
colder
temperatures.
The
effect
of
Sodium-
thiosulfate
and
EDTA
at
different
temperatures
is
also
discussed.
II
Tyrosinase
was
isolated
and
assayed
by
measuring
the
formation
of
Dopa-chrome
from
DL-Dopa
at
pH
6.8
an
37°C.
Tyrosinase
was
obtained
from
the
shrimp
liver
and
was
purified
by
approximately
25
times
its
activity
from
the
initial
extract
by
absorption
chromatography
through
Celite
545,
as
judged
by
the
rate
of
dopachrome
formation.
INTRODUCTION
Since
1923,
several
authors
(HAantsoN
4S:
HOOD
1923;
TANNER
1944;
GREEN
1949;
ALMEIDA
1955;
DEGKWITZ
1954;
BAILEY
1958;
KAKIMOTO
1956;
FIEGER
1956)
reported
on
the
black
discoloration
of
shrimp
and
suggested
various
methods
to
prevent
it.
Earlier
procedures
may
be
summarized
as
follows:
1
The
fresh
shrimp
'was
boiled
and
then
stored
at
low
temperature
(—
10°C
15°C)
;
2
The
fresh
shrimp
was
rapidly
freezed
and
stored
at
low
15°C)
;
3
The
fresh
shrimp's
head
was
taken
off
and
the
headless
shrimp
was
stored
at
low
tempera-
ture
(about
10°C)
;
4
The
fresh
shrimp
was
covered
with
ice
with
added
chemicals
or
dipped
in
cooled
chemical
solo
-
temperature
(about
10°C
This
work
was
accomplished
while
under
contract
as
Visiting
Researcher
at
the
Institute
OceanogrAilco
da
Universidade
de
Silo
Paulo,
on
a
Ford
Foundation
Grant.
Present
address:
Hokkaido
Kyoiku
University,
Hokkai-
do,
Japan.
(ions;
after
that
stored
at
low
temperature
5°C
10°C);
5
The
fresh
shrimp
was
stored
with
ice
with
added
antibiotic;
6
The
fresh
shrimp
was
freeze
dried
and
stored
at
Iow
temperature.
In
Brazil,
people
usually
prefer
to
consume
fresh
or
dried
shrimp.
Therefore,
it
seems
that
we
must
study
according
to
procedures
outlined
under
points
4,
5
and
6.
The
shrimp
landed
in
Santos
is
mainly
i
cama-
rao-sete-harbass,
and
i
carnariio-legitimo
s
.
The
amount
of
shrimp
landed
in
Santos
from
1962
to
1964
is
shown
in
Table
I.
As
shown
in
Table
I,
in
1964
the
total
land-
ings
of
shrimp
reached
about
13%
of
the
total
landings
of
fish
in
Santos.
Parallel
to
the
increase
of
the
catch
of
shrimp,
the
problem
of
the
black
spot
of
shrimp
has
become
increasingly
important
also
in
Brazil.
In
this
connection,
for
a
prevention
of
the
black
spot,
fishermen
have
already
used
chemicals,
called
Boim
Inst.
oceanogr.
S
Paulo,
16(1):1-11,
1967
1
TABLE
I
Landings
in
Santos
from
1962
to
1964
Year
Species
1962
Quantity
(kg)
1963
I
1964
Quantity
(kg)
l
Quantity
(kg)
'Ca
marao-sete-ba
rbas'
922,152
6
.8
%
1,194,639
6.3%
1,247,373
7.2%
'Ca
marlto-rosa'
604,997
4.5%
817,751
4.3%
891,022
5.1%
VamarAo-legitimo'
45,706
0
.3
%
34,133
0
.2
%
67,321
D.
4^.
Total
1,572,955
11.6
%
2,046,523
10.
8%
2,205,721
l
12.7
%
%
ratio
of
total
landings
'pO-de-camartio',
at
Santos,
they
are
sodium
sulfite
for
icamarao-rosar
and
at
Sao
Sehastiao,
boric
acid
for
'camarlio-sete-barbas'.
Other
chemicals
used
or
tested
in
other
coun-
tries
were
sodium
thiosulfate,
sodium
bisulfite
and
a
mixture
of
L
-ascorbic
and
citric
acids.
The
fishermen
of
U.S.A.
boats
fishing
along
the
Guyana
coast,
dip
shrimp
in
dilute
sodium
bisulfite
solution
at
low
temperature
for
a
few
minutes.
After
that,
they
cut
off
the
heads
and
stored
the
tails
at
low
temperature.
Moreover,
since
1940
many
researchers
have
studied
the
value
of
antibiotics
to
keep
the
quality
of
fish.
However,
from
the
view
point
of
food
hygiene,
the
Ministry
of
Health
and
Welfare
in
the
U.S.A.,
Canada
and
Japan
has
per-
mitted
fishermen
to
use
this
procedure
only
on
a
few
fishes
(salmon,
sea
trout
etc.).
On
shrimp,
DEGKW1TZ
(1954),
Fl
EGER
(1956),
GREEN
(1949),
SOUTHCOTT
et
al.
(1965)
and
MUCCIOLO
&
SCHNEIDER
(1965)
studied
the
effect
of
antibiotic
to
keep
quality
and
to
prevent
black
discoloration.
Except
for
the
result
of
Muccum_o
&
SCHNEIDER
(1965)
they
reported
that
antibiotics
kept
the
quality
of
shrimp
but
had
no
value
in
pre-
venting
black
spot
formation
which
is
caused
by
tyrosinase.
From
these
results
it
was
observed
that
chemi-
cals,
such
as
sodium
thiosulfate,
sodium
bisulfite
and
others
were
significant
in
preventing
the
black
spot
but
did
not
contribute
towards
keeping
the
quality
of
shrimp.
Therefore,
shrimp
treated
with
chemicals
should
be
stored
at
low
temperature.
It
seems
that
the
black
spot
of
shrimp
is
caused
by
the
action
of
the
contained
enzyme
(tyrosinase)
and
of
contaminating
bacteria.
Therefore,
for
a
study
of
this
problem,
the
author
separated
it
into
two
parts:
a
study
of
the
contained
enzyme
and
a
study
of
the
contaminating
bacteria.
The
effect
of
chemicals
in
preventing
discolo-
ration
by
tyrosinase
in
shrimp
was
studied.
It
was
observed
again
that
reducing
reagents
were
useful
in
preventing
it.
Furthermore,
it
was
observed
that
a
chelate
reagent,
such
as
EDTA,
was
similarly
va-
luable
as
a
reducing
agent.
The
tyrosinase
of
shrimp
was
also
studied.
Bo
-
DINE
&
ALLEN
(1941)
reported
on
the
presence
of
protyrosinase
in
blood
of
tray
-fish
and
KAKIMOTO
(1956)
on
the
tyrosinase
in
blood,
liver
and
other
organs
of
Japanese
spiny
lobster.
However,
no
pur-
ified
enzyme
has
hitherto
been
obtained
from
marine
animals.
The
author
has
undertaken
the
isolation
and
purification
of
the
tyrosinase
of
'camarao-sete-barbas'
for
a
study
of
its
properties
and
obtained
the
enzyme
purified
approximately
25
-times
specific
activity.
In
the
present
paper,
the
experimental
results
on
the
effect
of
chemicals
in
preventing
black
spot
caused
by
a
tyrosinase
and
a
method
of
the
isolation
and
purification
of
enzyme
from
the
'camarao-sete-bar-
bas'
liver
are
described.
EXPERIMENTAL
PROCEDURE
I.
On
the
effect
of
some
chemical
reagents
on
the
black
spot
of
shrimp
MATERIAL
'Camario-sete-barbas'
(Xyphope-
naeus
kroyeri)
samples
were
taken
at
the
offing
of
Santos,
from
March
to
November,
1965.
'Camarilo-
rose
(Penaeus
aztecus)
was
bought
at
the
central
market
of
Santos.
CHEMICALS
Sodium
sulfate,
L
-ascorbic
acid,
Sodium
thiosulfate,
Ethylendiamin
tetra
acetic
(EDTA),
Acetic
acid,
n-Buthanol,
Phenol,
Acetone
and
Ninhydrine
were
used.
All
reagents
were
Merck
A.G.
Organoleptic
tests
were
done
in
two
methods:
A
Chemicals
were
added
directly
on
shrimp
and
the
treated
shrimp
were
stored
at
low
temperature.
That
is,
at
first,
shrimp
were
separated
into
three
masses
(I,
II
and
III).
Next,
each
mass
was
separ-
ated
into
three
or
five
groups
containing
10
or
20
shrimps.
Then,
each
chemical
(one
of
tenth
of
weight
of
shrimp)
was
added
to
each
mass.
That
is,
sodium
sulfate
was
added
to
mass
I,
L
-ascorbic
acid
to
mass
II
and
EDTA
to
mass
III,
respectively.
Then,
shrimp
treated
were
stored
in
the
electric
re-
frigerator
at
0°C
r
2°C.
B
Shrimp
were
clipped
in
each
cooled
chemical
solution
for
30
mi-
nutes
at
low
temperature
and
then
stored
at
low
temperature.
That
is,
shrimp
were
separated
on
the
same
way
as
described
above,
and
then,
each
mass
was
dipped
in
each
cooled
chemical
solution
pre
-
Holm
Inst.
oceanogr.
S
Paulo,
16(1):1-11,
1967
2
pared
at
0°C
•-•-•
2°C.
for
30
minutes.
After
that,
shrimp
were
taken
out
from
each
solution
and
stored
in
the
electric
refrigerator
at
0°C
2°C
and
5°C
10°C.
The
concentration
of
various
chemicals
solution
in
shown
in
Table
IL
TABLE
II
Concentration
of
chemical
solutions
Chemicals
Concentration
(5,
t
Na
r
SO
4
a
I.
Ascorbic
acid
1
Na
2
SA
a
EDTA
0.5
EDTA
1
EDTA
1.25
EDTA
2.5
EDTA
5
After
the
procedure
described
above,
daily
observations
were
made
to
see
whether
the
shrimp
in
each
group
became
black
or
not.
CnEmicat.
TEST
a)
The
change
of
the
free
amino
acids
content
in
muscle
of
shrimp
during
storage
at
0°C
2°C
was
observed
by
means
of
paper
chromatography.
Preparation
of
sample
for
the
paper
chromato-
graphy
One
gram
of
muscle
was
homogeneized
for
a
few
minutes
with
10
volumes
of
water.
After
10
minutes,
5
ml
of
20%
trichloracetic
acid
were
added
and
the
precipitate
was
filtered
off.
The
fil-
trate
obtained
was
dried
in
a
vacuum
dessircator,
then,
0.1
ml
of
warm
water
(60°C)
was
added
and
the
precipitate
was
completely
dissolved.
At
first,
one
third
volume
of
the
solution
was
applied
to
one
dimension
paper
chromatography.
Two
third
volu-
mes
of
the
remaining
solution
were
applied
to
two
dimension
paper
chromatography
at
room
tempera-
ture
(about
25°C).
Solvent
system.
n-Buthanot
acetic
water
(4:1:5
sup.)
ai.d
phenol
water
(4:1)
were
used.
acid
Development
of
color
0.25%
ninhydrin
ace-
tone
solution
and
special
reagent
for
a
few
amino
acids
(isatin
for
praline,
Pauly
test
for
histidine
and
tyrosinase
and
Sakaguchi
test
for
arginine)
were
used.
b)
pH
was
measured
with
Metrohm
A
C
Heri-
sou
Type
E
148
C.
II.
On
the
isolation
and
purification
of
tyrosinase
from
shrimp
liver
MATERIAL
Shrimp
('camariio-sete-barbas
i
,
Xyphopenaeus
kroyeri)
was
captured
at
the
offing
of
Santos,
from
March
to
November,
1965.
All
reagents
used
were
analytical
grade,
unless
other-
wise
indicated.
Celite
545
was
washed
several
times
by
suspending
it
in
distilled
water
and
decanting.
The
slurry
was
poured
into
the
column
and
equili-
brated
with
0.005
M
sodium
phosphate
buffer,
at
pH
7.2
by
running
several
hold-up volumes
of
the
buffer
through
each
column.
Sephadex
G-50
was
prepared
in
the
similar
way
as
the
Celite
545
except
that
0.01
M
sodium
phosphate
buffer
was
used
for
washing
and
equilibration.
Enzyme
assay
Tyrosinase
was
assayed
by
measuring
the
formation
of
2-carboxy-2,3-dihydroin-
do1-5,6-qu
i
none
(
Dopa-c
hrome
)
from
3,4
-di
hydroxy-
DL-phenylalanine
(DL-dopa)
at
pH
6.8
and
at
37°C.
The
assay
system
consisted
of
0.2
nil
of
enzyme
extract
preparation,
1.8
ml
of
0.1
M
sodium
phos-
phate
buffer
at
pH
6.8,
and
0.1
mg
of
DL-dopa
dissolved
in
5
ml
of
the
same
buffer.
The
forma-
tion
of
dope
-chrome
at
37°C
was
measured
calori-
metrically
using
a
Bausch
&
Lomb
Specironic
20
at
480
my..
Tyrosinase
unit
According
to
the
method
of
HAROWITZ
(1960)
and
FLING
(1963),
an
amount
of
enzyme
which
produces
an
absorbency
increase
of
0.1
at
480
mp.
in
the
first
5
minutes,
was
adopted
as
a
unit
for
the
tyrosinase.
Protein.
determination
During
the
course
of
the
isolation,
protein
concentration
was
measured
colorimetrically
by
the
biuret
method
using
a
Bausch
&
Lomb
Spectronic
20
at
560
mil
or
750
miz.
RESULTS
I.
On
the
effect
of
some
chemical
reagents
on
the
black
spot
of
shrimp
The
results
of
organoleptie
methods
are
shown
in
Tables
The
value
indicated
in
the
Tables
are
shown
as
a
ratio
of
blackening
against
total
shrimp.
Table
III
shows
the
results
of
method
A.
From
the
results
of
Table
HI,
L-arcorhic
acid
was
indicated
as
having
a
better
effect
than
others,
hut
in
this
case,
the
surface
of
a
few
shrimp
were
TABLE
III
Discoloration
during
storage
(powder)
at
0°C
2°C
Storage
period
days
Reference
Chemicals
Na.
50
.,
%
L-Ascorbic
acid
%
EDTA
%
0
0
0 0 0
1
15
0
0
0
2
25
20
0
33
3
70
WI
0
40
4
30
0
40
5
/
/
/
G
60
20*
50
7
80*
to
become
yellow
13olm
Inst.
oceanogr.
S
Paulo,
16(1):I-11,
1967
3
covered
with
a
yellow
color
(that
is
yellow
discolo-
ration)
instead
of
blackening.
The
results
of
method
B
are
shown
in
Tables
IV,
V
and
VI.
Tables
IV
and
V
show
the
results
for
'camarito-sete.barbas'
when
storage
temperature
was
0°C
2°C
(Tab.
IV)
and
5°C
10°C
(Tab.
V),
respectively.
EDTA
solution
was
indicated
as
having
a
effect
than
others.
Table
VI
show
the
results
for
'camario-
rosai.
From
the
results
of
Table
VI,
it
is
evident
that
L
-ascorbic
acid
strongly
delayed
black
spot
forma-
tion.
Sodium
thiosulfate
and
EDTA
delayed
black
TABLE
IV
Discoloration
during
storage
of
d
camariia-sete-barbas'
(solution)
at
0°C
—2°C
Storage
period
days
Reference
Chemleals
L
-Ascorbic
acid
%
I
EL/TA
%
I
0.5%
1.25%
J
2.5%
I
5.0%
0
0
0
0
1
0 0
0
0
0
0
2
15
0
10
0
10
3
40
10
20
10
20
4
55
40
20
20
60
40
5
100
I
6
50
30
40
60
50
TABLE
V
Discoloration
during
storage
of
'camarao-sete-barbas'
(solution)
at
—5°C
—10°C
Storage
period
days
Reference
%
Chemicals
Na,
SO,
I
%
L
-Ascorbic
acid
%
EDTA
%
0
0
0
0
0
1
0
0
0
0
2
0
0
0
0
3
0
0
0
0
4
0
0
0
0
5
0
u
0 0
6
0
0
0
0
7
10
0
0
0
8
20
0
0
0
9
20
10
0
0
10
20
10
0
0
11
/ /
/
/
12
/
/
13
50
20
10
10
From
the
results
of
Tables
IV
and
V,
it
was
observed
that
the
formation
of
black
spot
was
strong-
ly
delayed
at
low
temperature.
In
this
case,
shrimp
didn't
become
black
for
about
6
clays
without
chemi-
cals,
but
after
that,
the
black
spot
appeared
rapidly
on
the
shrimps.
The
author
was
unable
to
indicate
organoleptic
difference
between
these
chemicals
up
to
8
days,
but
he
could
indicate
the
differences
between
three
chemicals
from
pH
value
measured
at
8'
days.
The
results
of
the
pH
value
were
shown
in
Table
VII.
At
high
temperature
(0°C
2°C),
diluted
TABLE
VI
Discoloration
during
storage
of
'camatto-rosa'
(solution)
at
—5°C
Storage
period
days
Reference
"A
Chemicals
Na
:
S.,0
3
%I
Na,SO
4
%
acid
5,
L-Ascorb
ic
5,
EDTA
0
0
0
0
0
0
1
0
0
10
0
0
2
20
10
20
0
10
3
60
10
50
0
10
10
50
0
10
5
10
50
0
10
6
10
50
0
10
7
20
60
0
20
20
70
0
20
9
40
80
0
40
spot
formation
too,
but
their
effects
were
not
as
strong
as
',ascorbic
acid,
while,
sodium
sulfate
had
hardly
any
value
in
preventing
black
spot
formation.
At
the
9th
day,
pH
value
of
shrimp,
which
showed
no
black
spot
up
to
that
day,
was
measured
and
the
results
obtained
are
shown
in
Table
VII.
The
author
was
able
to
indicate
slight
differences
between
three
chemicals,
except
sodium
sulfate.
The
results
of
the
chemical
test
are
shown
in
Figures
1-5.
Rolm
Inst.
oceanogr.
S
Paulo,
16(1):1-11,
1967
4
TABLE
VII
-
pH
value
of
each
fraction
during
storage
‘Camallo-sete-barbas'
'Camarito-roses'
pH
value
Chemicals
-
5°C
,-...
-10
0
C
0
0
C
,...
-
2°C
-
5°C
....
-10
0
C
81h
days
fter
bla
A
ckening
6th
days
After
blackening
9th
days
L
-Ascorbic
acid
6.80
7.70
7.40
7.70
7.11
0.5
%
EDTA
-
-
7.20
7.80
-
c
1.25
%
EDTA
7.20
7.80
7.50
7.50
7.38
(1)
7.1
0
-
6
.
2.5
%
EDTA
-
--
-
7.80
-
W
5.0
%
EDTA
-
-
-
8.00
-
Na
t
S0,
7.50
8.00
8.00
8.00
7.59
Na,S.0,
-
_
-
-
7.39
Na,S0.
-
-
7.50
8.20
-
...
.6
1
L
-Ascorbic
3
o
acid
- -
6.80
5.00
(2)
-
a.
EDTA
-
-
6.20
6.70
-
1)
Concentration
of
EDTA
Is
1
2)
Yellow
color
Figure
1
shows
the
results
of
one
dimension
paper
chromatography.
3C
i
a
^.
:-
-)
0
o
eif
, 0
Q.1
c2)
ac,
e
c,
e4
0
24
36
48
h
Tyr
Fig.
1 -
One
dimension
paper
chromatogram
of
tyrosinase
and
shrimp
muscle
after
0,
24,
36
and
48
hours
storage
time
at
0
0
C
to
-2°C.
Figue
2
shows
the
results
of
fresh
material.
Figure
3
shows
the
results
after
24
hours
of
storage.
Figure
4
shows
the
results
after
36
hours.
Figure
5
shows
.the
results
after
48
hours.
After
48
hours,
n-
BUT
liANOL
7
r.
C\
Fig.
2
-
Two
PHENOL=
WATER
(4=1)
...
dimension
paper
chromatogram
of
fresh
shrimp
muscle.
the
surface
of
shrimp's
neck
became
slightly
black.
From
Figures
2.
3,
4
and
5,
the
author
was
able
to
indicate
that
some
amino
acids
(pro.
arg.
tyr.
-
amino
butyric
acid
etc)
has
disappeared
and
an
unknown
substance
was
produced
during
storage
at
0°C
-
2°C.
The
results
of
the
change
of
free
amino
acids
in
the
muscle
of
shrimps
are
shown
in
Table
VIII.
Bolm
Inst.
oceanogr.
S
Paulo,
16(1):1-11,
1967
5
n
-
BUTHANOL
=
ACETIC
ACID=
WATER
(4:1:5).„
PHENOL
=
WATER
(4=
1)
Fig.
3
Two
dimension
paper
chromatogram
of
shrimp
muscle
after
24
hours
of
storage
time
at
0°C
to
—2°C.
or
n-
BUTHANOL
=
ACE
TIC
ACID
=
a
PHENOL=
WATER
(4
:1)
Fig.
4
Two
dimension
paper
chromatogram
of
shrimp
muscle
after
36
hours
storage
time
at
to
—2°C.
The
author
again
measured
the
pH
as
an
indi-
cator
of
autolysis.
These
results
are
shown
in
Table
IX.
cr
I
-
iU
n
-
BUTHANOL
=
ACE
TIC
ACID
PHENOL=
WATER
(4
:
1)
Fig.
5
Two
dimension
paper
chromatogram
of
shrimp
muscle
after
48
hours
storage
time
at
0°C
to
—2°C.
I I
.
On
the
isolation
and
purification
of
the
enzyme
related
to
the
formation
of
the
black
spot
of
shrimp
A
summary
of
the
isolation
procedure
of
tyro-
sinase
from
shrimp
liver
is
shown
in
Figure
6
and
Table
X.
All
the
steps
were
carried
out
below
10°C.
Unless
otherwise
indicated
the
buffer
referred
to
be-
low
is
0.1
M
sodium
phosphate
at
pH
7.2.
Extration
Fresh
liver
of
shrimp
was
mixed
with
two
volumes
of
cooled
buffer
and
allowed
to
stand
for
21
hours;
then
the
extract
was
filtered.
Preliminary
purification
The
filtrate
was
treated
with
50
ml
of
0.2
M
Mauganous
sulphate
per
liter.
The
pH
value
of
this
solution
has
to
be
kept
at
7.2.
After
standing
for
24
hours,
the
preci-
pitate
was
removed
by
centrifugation
and
washed
once
with
chilled
water.
Ammonium
sulphate
precipitation
To
the
mixture
of
the
supernatant
and
the
washing,
solid
ammonium
sulphate
was
slowly
added
by
stirring,
to
bring
the
concentration
of
ammonium
sulphate
to
60%,
and
then
allowed
to
stand
overnight.
The
precipitated
protein,
containing
the
tyrosinase,
was
TABLE
VIII
Change
of
free
amino
acid
in
the
muscles
of
'camarao-sete-barbas'
Amino
acid
Phe
Leu
Val
+
Met
Try
Tyr
et
-Amino
-
butyric
acid
Pro
Ala
A
rg
Thr
Ser
Gly
Glu
Asp
0
24
36
+
+
+
+
+
+
+
+
+
+
.4-
_
±
+
+
±
+
+
7
+
±
++
-I-
-I-
+
+++
+
+
±
+
+
+
+
+
-I-
++
+
-i
-
+
+
+
+
+
+
Beim
Inst.
oceanogr.
s
Paulo,
16(1):1-11,
1967
6
TABLE
IX
-
Change
of
pH
value
during
storage
Storage
period
h.
12
24
36
48
Storage
temperature
pit
value
7.0
7.0
6.9
7.3
6.8
7.0
8.2
7.2
(-
2°C)
(-
2°C)
TABLE
X
-
Purification
of
tyrosinase
Fraction
Volume
Total
units
Total
protein
Specific
activity
Purification
ml
DOPA
mg
DOPA
unit/
mg
Crude
extract
200
200.000
15
13.33
1
MnSO,
supernatant
300
207.480
12.9
16.08
1.2
First
ammonium
sulfate
precipitate
100
130.802
3.8
34.42
2.6
Extract
of
acetone
precipitate
40
118.700
1.0
118.70
5.9
Solution
passed
on
104.160
0.64
162.75
12.2
Sephadex
column
20
Celite
eluate
50
16.670
0.0536
329.88
24.9
FRESH
LIVER
Ft
LTRATE
FILTRATE
PRECIPITATE
PRECIPITATE
PRECIPITATE
add,
2
volumes
of
0.1
M
sodium
phosphate
buffer,
pH
6.8
in
the
low
temperature,
after
24
hours,
filtered.
add.
0.2
M
Mn-SO.
(50
m1/1)
keep
pH
7.2,
after
24
hours,
filtered.
add.
solid
(NH,),
SO,,
at
0
°
C,
after
24
hours,
the
precipitate
was
collected
by
centrifugation
at
2,000
r.p.m.
for
30
minutes.
dissolved
in
0.1
M
sodium
phosphate
buffer
pH
7.2,
add.
2
volumes
of
acetone
at
-10°C
the
precipitate
was
collected
by
centrifugation.
extraction
with
small
quantities
of
buffer,
add.
solid
(NH.).
SO,,
the
precipitate
was
collected
by
centrifugation.
dissolved
in
buffer,
passed
on
Sephadex
G-50
column,
add.
equal
volume
of
chilled
water.
CE
LITE
CH
ROmATOGP,APHY
Fig.
6
-
isolation
❑f
enzyme
from
'camarao-sete-barbas
.
.
collected
by
centrifugation
at
2.000
r.p.m.
for
30
minutes.
Acetone
precipitation.
-
The
precipitate
was
dissolved
in
a
minimal
amount
of
buffer
and
cooled
to
-
10°C,
then
two
volumes
of
acetone
were
added
slowly
by
stirring.
The
suspension
was
centrifuged,
the
supernatant
of
which
was
discarded.
The
tyro-
sinase
was
taken
out
of
the
precipitate
by
extracting
it
several
times
with
small
amounts
of
buffer
and
was
precipitated
from
the
combined
extracts
by
addition
of
solid
ammonium
sulphate.
Procedure
on
Sephadex
G-50
-
The
ammonium
sulphate
precipitate
from
the
preceding
step
was
dissolved
in
buffer.
The
solution
was
made
free
from
ammonium
sulphate
by
passing
it
through
Sephadex
G-50
column
(2.5
x
8
or
23
x
15
cm)
which
had
been
equilibrated
with
0.01
M
buffer.
Celite
chromatography
-
The
ammonium
-free
eluate
from
the
preceding
was
diluted
with
two
volumes
of
water
and
placed
❑n
a
column
(
2.5
x
8
or
2.5
x
15
cm)
of
Celite
54.5
which
had
been
equilibrated
with
❑.005
M
buffer.
Stepwise
elution
of
the
column
was
employed
for
the
chromatographic
separation
of
the
enzyme.
The
effluent
volume
of
each
tube
was
controlled
for
5
ml
in
30
minutes.
About
70%
of
the
activity
originally
placed
on
the
column
was
recovered
in
the
0.02
M
buffer.
These
results
are
shown
in
Figure
7.
DISCUSSION
The
author
studied
the
effect
of
chemicals
against
shrimp
blackening
with
organoleptic
and
chemical
methods.
Fishermen
except
for
those
operating
from
Sao
Sebastiao
did
not
use
chemicals
for
the
prevention
of
black
spot
formation
in
'camarao-sete•barbas'.
Therefore
is
valuable
t❑
use
icamargo-sete-barbas'
for
studies
in
the
laboratory
on
the
effect
of
chemi-
cals
on
the
prevention
of
the
black
spot
formation.
From
the
results
of
Table
III
and
V,
the
author
observed
that
L
-ascorbic
acid
had
very
strong
value
in
preventing
black
spot
formation.
Balm
Inst.
oceanogr.
S
Paulo,
Hill/A
-
lit
196
7
7
0,
00
-
0.500
-
02
M
0
05
0.1
0.2
M
50
100
150
mi.
ENZYME
OPTfCAL
DENSITY
0.02
M
0.05
❑.1
NI
0.2
NI
50
PROTEIN
Fig.
7
Elution
of
tyrosinase
from
Celite
545
column.
If
the
phenomenon
of
blackening
is
caused
by
the
action
of
tyrosinase
in
shrimp,
it
should
be
inhibited
when
copper
ion
is
removed
from
the
shrimp
muscles,
because
it
is
subject
to
activation
by
tyrosinase.
It
is
known
that
copper
ions
are
contained
in
the
muscles
of
shrimp,
therefore
a
chelate
reagent
(EDTA)
was
used
to
remove
it.
And
it
was
observed
that
the
effect
of
EDTA
was
much
the
same
as
that
of
L
-ascorbic
acid
when
dilute
solution
of
EDTA
was
used.
iCamarao-rosa
r
treated
with
chemicals
(sodium
sulfate)
were
used.
From
Table
VI,
the
author
observed
that
L
-ascorbic
acid,
sodium
thiosulfate
and
EDTA
were
valuable
in
preventing
black
spot
for-
mation
caused
by
tyrosinase.
L
-ascorbic
acid
and
sodium
thiosulfate
were
reducing
reagents
and
strongly
delayed
black
spot
formation.
The
fact
agreed
well
with
the
results
of
other
authors.
Sodium
thiosulfate.
sodium
sulfate
and
sodium
bisulfite
have
various
uses,
are
widely
used
and
their
price
is
reduced.
But,
when
we
use
these
rea-
gents,
we
must
keep
within
the
limits
allowed
by
legislation
on
the
hygenie
of
food.
While
L
-ascorbic
acid
and
EDTA
are
harmless
their
price
is
high.
The
author
thinks
that
this
is
the
difficulty
to
the
widespread
adoption
of
these
chemicals
as
preser-
vatives.
In
this
experiment,
the
author
observed
that
reducing
reagents
and
chelate
reagents
are
valuable
in
preventing
black
spot
formation
in
shrimp.
From
these
results,
the
author
is
also
able
to
conclude
that
blackening
occurs
by
the
action
of
tyrosinase
in
shrimp.
The
author
recommends
the
following
method
for
the
prevention
of
the
black
spot
formation
of
shrimp
as
follows:
Procedure
The
fresh
shrimp
are
dipped
in
the
cooled
chemical
(reducing
reagent
or
EDTA)
solution
for
a
few
minutes.
After
that,
treated
shrimp
are
put
in
a
box
and
stored
at
low
temperature
for
100
150
ml
the
prevention
of
the
evaporation
of
moisture
from
shrimp
and
to
inhibit
the
action
of
contaminated
bacteria.
Moreover,
the
author
studied
the
change
of
the
free
amino
acids
in
shrimp
muscles
when
stored
at
about
2°C.
At
this
temperature,
black
spot
appeared
on
the
surface
of
the
shrimp
within
48
hours.
The
fresh
shrimp
have
many
free
amino
acids
in
the
muscle
as
shown
in
Figure
2.
This
fact
agreed
well
with
the
results
of
ALMEIDA
(1954)
and
CAMIER
et
al.
(1951).
On
paper
chromatogram,
the
author
did
not
identify
some
substances
that
would
have
occurred
during
storage.
As
shown
in
Table
IX
it
was
observed
that
autolysis
occurred
in
shrimp.
The
author
was
unable
to
clear
the
relation
of
blackening
and
free
amino
acids
in
muscle,
but
he
thinks
that
part
of
amino
acids
wilI
be
used
as
substrate
of
tyrosinase,
because
tyrosinase
and
tryptophane
dis-
appeared
at
once.
Moreover,
the
author
studied
the
isolation
and
purification
of
the
enzyme
related
to
the
discolora-
tion
of
shrimp.
In
our
preparatory
experiment,
it
was
studied
wheter
tyrosinase
present
or
not
in
various
organs
of
icamariio-sete-barbas'
and
on
some
solvents
for
the
extraction
of
tyrosinase
from
the
'camarlio-sete-bar-
has'
liver.
The
results
obtained
were
shown
in
Table
XI.
From
the
results
of
Table
XI,
tyrosinase
was
extracted
from
'camariio-sete-barbas'
liver
with
phos-
phate
buffer,
at
pH
6.8
7.2.
Also
the
activity
of
tyrosinase
of
three
sorts
of
shrimp
(icamariio-sete-
barbs?,
'camariio-rosa!
and
'camariio-legitimo')
was
compared.
The
results
obtained
were
shown
in
Fi-
gures
8
and
9.
From
the
results
shown
on
Figures
8
a,
b,
and
9,
it
may
be
seen
that
the
activity
of
tyrosinase
from
these
shrimps
indicated
the
same
activity,
after
24
hours.
13olm
Inst.
oceanogr.
S
Paulo,
10
(1
):1-11,
1967
8
TABLE
XI
The
enzymatic
activity
of
the
various
organs
from
'camarifto-sete-barbas'
Hour
Sample
0.5
1
1.5
2
3
5
8
15
24
tyr
Y
DB
+ +
++ +++
R
lip
+ +
A
tyr
+ + + ±
++
-k+
+++ +++
Q
Hp
±
±
+
+
+
++
tyr
Y
DR
DV
+ + +
R
Hp
±
±
B
tyr
+ + + + +
+ +
+
Q
Hp
±
±
tyr
RV DR
DR
+
+
+
+ + +
+
R
Hp
+
+
C
tyr
+ + + +
+
r
-
t
+
+++
Q
Hp
_
_
_
+
R
tyr
Hp
± ±
±
+
±
±
±
+
--
+
+
1)
tyr
± ±
±
±
± ±
± + +
Q
1-1,0
R
14
,0
E
.
.... ....
.
.
tyr
--
_
Q
Hp
_
_
tyr
_ _
R
Hp
--
F
lip
".
@
tyr
_
Sample
A
extract
solution
by
physiological
salt.
B
extract
solution
by
phosphate
buffer,
pH
6.8.
C
extract
solution
by
30%
acetone.
D
liver;
E
muscle
and
F
intestine,
D,
E
and
F
were
extract
solution
by
phosphate
buffer,
pH
6.8.
R
=
raw
material;
Q
=
heated
material;
Y
=
yellow;
DB
=
dark
brown;
DR
=
dark
red;
DV
-=
dark
violet;
RV
=
redIsh
violet;
+,
black
(darkeing),
—,
no
black.
As
shown
in
Table
X,
the
tyrosinase
was
purifi-
ed,
by
approximately
25
times
its
activity
from
the
initial
extract,
by
means
of
absorption
chromato-
graphy
through
Celite
545,
as
judged
by
the
rate
of
the
formation
of
dopachrome.
Recently,
the
purification
of
tyrosinase
from
various
sources
such
as
mushroom,
hamster
and
Nett-
rospora
was
published
(HAFtovvaz
1960;
FLING
1963;
BoucHn.toux
et
at.
1963;
POMERANTZ
1963).
The
author
was
unable
to
compare
his
results
with
the
present
ones,
because
of
the
lack
of
more
detailed
data
on
the
physical
and
chemical
properties
of
this
enzyme,
which
will
be
reported
elsewhere.
Balm
Inst.
oeeanogr.
S
Paulo,
16(1):1-11,
1967
9
1.0
0
Q5
00
N
cr
a
0
iASD
0
0
0
-
A
Fig.
R
Enzyme
0
)90541.
0
0
241
>SETE
BARBAS
A
0.
er,
Dopo
as
substrate
Tyrosin
as
substrate
activity
of
'camarAo-sete-barbas'
and
`camarBo-rosa'.
OOPA
I
AS
SUBSTRATE
TYROSINE
}AS
SUBSTRATE
ti---4/
O
ROSA
0
46
A
•LEG1TiNtO
400
500
600
Fig.
9
Enzyme
activity
of
'camarto-rasa'
and
'carnarAo-legitlmo%
ACKNOWLEDGEMENTS
The
author
wishes
to
express
his
sincere
thanks
to
Dra.
M.
VANNUCCI
for
her
support
and
aIso
wishes
to
thank
Miss
DULCINDA
RODRIGUES
DA
SILVA
and
Mr.
Lutz
SANCHES
for
their
helpful
assistance.
RESUMO
Foram
estudadas,
no
presente
trabalho,
as
causas
do
enegrecimento
do
camera°,
tendo-se
verificado
que
o
mesmo
a
causado,
fundamentalmente,
pela
aciio
da
tirosinase,
produzida
no
hepato
pancreas
do
crusta-
ceo.
isolamento
e
a
purificacao
da
enzinta
foram
realizados.
A
purificacao
foi
feita,
ate
se
obter
uma
atividade
25
vexes
maior
do
que
o
extrato
initial,
par
meio
de
cromatografia
de
absorgao,
atraves
de
Celite
545
e
avaliada
atraves
da
velocidade
de
for-
macao
de
dopacromo.
use
de
diferentes
drogas
tamhem
foi
tratado
experimentalmente,
tendo-se
verificado
que
o
melhor
inibidor
do
processo
de
enegrecimento
é
o
acid°
L-arcarbico
que
evita
a
formacao
da
mancha
preta
durante
9
dias
a
temperatura
de
0
a—
2°C
e
du-
rante
tempo
mais
longo
em
temperaturas
mais
bai-
xas.
Tiosulfato
de
soclio
c
EDTA
tambem
tern
boa
agar)
inibidora.
As
tecnicas
empregadas
sac)
deseri-
tas
detalhadamente
e
e
apresentada
discussao
corn
resultados
obtidos
por
outros
autores.
Ao
que
se
sabe,
esta
4
aprimeira
vez
que
tirosinase
6
isolada
de
urn
invertebrado.
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W.
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1963.
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)
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1967
11