Values of protease activity showed a great variation within species, from 37
units/g fresh viscera in the blue ling (
Molva dypterygia
) to 278 units/g fresh
viscera in the European pilchard (
Sardina pilchardus)
. The same variation was
observedwhenactivitywas expressed in relation to soluble protein, with values
ranging from 12 units/mg soluble protein measured in the Argentine hake
(
Merluccius hubbsi)
to more than 400 units/mg soluble protein in haddock
(
Melanogrammus aeglefinus).
The absence of correlation between both values
reflects that the concentration of active enzyme in visceral samples is affected
by a great number of factors (species, feeding status, procedures used for
processing and storing of tissues, etc.). After this preliminary screening, some
of the species were not considered in further steps of the study due to the
difficulties to obtain samples in amount enough, or stored under the required
conditions. This aspect was related both to the size of catches for a species
and to the procedures followed in the vessels for evisceration of fish.
Preparation of active and stabilized products
The process followed in order to obtain a Purified Enzyme Extract (PEE) was
characterized by three main stages; extraction, purification and stabilization,
Frozen viscera
Freezer -80ºC
Weight of viscera with scales
Homogenization or crushing of viscera
(with homogenization potter)
Whole process is made keeping
temperature conditions and using the pH
8 buffer.
The homogenized sample is distributed in centrifuge tubes
and is balanced in a centrifuge
Centrifugation at 4ºC for 20 min and at 20,000 rpm
Precipitation of the higher liquid phase (supernatant) with ammonium
sulphate (30%)
It is made on a permanent agitation basis and at 4ºC for 30 minutes
Sample filtration to remove impurities or
solid waste
Balancing of the sample in centrifuge tubes
and centrifugation at 4ºC for 30 min and
15,000 rpm
Filtration of the supernatant
Dialysis of the supernantant during 24 h
changing twice the dialysis buffer.
Freezing of the dialyzed
product
Lyophilization of the sample
1:4 (w/v)
homogenization buffer
1ª
Stage
2ª
Stage
3ª
Stage
4ª
Stage
5ª
Stage
6ª
Stage
7ª
Stage
68
Use of purified extracts from fish viscera as an enzyme additive in feeds for juvenile marine fish
Figure 2.
PEE extraction,
purification and
stabilization
1...,60,61,62,63,64,65,66,67,68,69 71,72,73,74,75,76,77,78,79,80,...84