Value added fish by-products - Nordic Innovation

2.3.1.11 Partial purification of the CGRP-like molecules
The CGRP-like molecules included in the hydrolysates obtained from whole frozen and
minced fresh samples in post-rigor state (ten min of hydrolysis) were pre-purified by gel
exclusion chromatography on a HW 40 toyopearl column (2.5 x 33.5 cm) using ammonium
acetate 0.2 M, pH 5 as eluant. The flow rate was 22 ml/hour. The column was calibrated with
the following molecular weight markers: aprotinin (6000 Da), CGRP (3750 Da), and
bacitracin (1411 Da). Aliquots were analyzed for CGRP-immunoreactivity. Immunoreactive
fractions were then analyzed using CGRP radioreceptorassay.
2.4 Comparison of chemical and functional properties of lab made and
commercial available fish powders
2.4.1 All chemical and functional analysis as described above.
2.4.2 Molecular weight distribution
Dry powder was dissolved in 50 mmol/l buffered imidazole solution at pH 7.0 (100 mg/ml).
The sample was examined using a Superdex Peptide 75 10/300GL column with an Akta
FPLC academic edition. Detection wavelength was set to 280 nm.
2.4.3 Amount and composition of free amino acids
Amount of free amino acids was determined by high-pressure liquid chromatography
(HPLC). Dry powders were dissolved in 0.05 M phosphate buffer (pH=7.0) and centrifuged
for 10 minutes at 10 000 rpm. Reversed phase HPLC by precolumn fluorescence
derivatization with o-phthaldialdehyde (SIL-9A Auto Injector, LC-9A Liquid Chromatograph,
RF-530 Fluorescence HPLC Monitor, all parts from Shimadzu Corporation, Japan) was
performed using a NovaPak C18 cartridge (Waters, Milford, MA, USA). Glycine/arginine
and methionine/tryptophane were determined together, as their peaks merged. This analysis
was performed twice on each sample.
2.4.4 Amount and composition of total amino acids
The amino acid composition of powdered samples was determined by digestion in 6 M HCl at
105 o C for 22 h followed by neutralisation of hydrolysates. After dilution and filtration
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