Fish scale collagen. Preparation and partial characterization. Summary Fish scale was decalcified and disaggregated and then collagen was prepared by limited pepsin digestion. The yields of collagens were very high on a dry weight basis; sardine trimers with a chain composition of (a1)2a2. Although the denaturation temperature of the collagen was lower than land animal collagen, fish scales will have potential as an important collagen source for use in various industries.
Trang 1Fish scale collagen Preparation and partial
characterization
Takeshi Nagai,1* Masami Izumi2& Masahide Ishii3
1 Department of Food Science and Technology, National Fisheries University, Shimonoseki, Yamaguchi 7596595, Japan
2 Ribro Com, Inc., 1-5-10 Nishi-shinbashi, Minato-ku, Tokyo 1050003, Japan
3 Staff Labbi, 6-6-28 Akasaka Minato-ku, Tokyo 1070052, Japan
(Received 25 October 2002; Accepted in revised form 30 June 2003)
Summary Fish scale was decalcified and disaggregated and then collagen was prepared by limited
pepsin digestion The yields of collagens were very high on a dry weight basis; sardine
50.9%, red sea bream 37.5% and Japanese sea bass 41.0%, respectively These scale
collagens were heterotrimers with a chain composition of (a1)2a2 Although the
denatur-ation temperature of the collagen was lower than land animal collagen, fish scales will have
potential as an important collagen source for use in various industries
Keywords Alternative source of collagen from cattle skin, underutilized resources, yield.
Introduction
Collagen is the protein that is found in the highest
concentration, about 30%, in the living body The
main sources of industrial collagen are limited to
those from bovine and pig skin and bones
However, the existence of bovines infected with
Bovine Spongiform Encephalopathy (BSE) has
been reported in Japan (Yamauchi, 2002) It
becomes a matter of great important to solve the
problems created by BSE One alternative is to
replace bovine collagen with another source As
part of a study looking at the effective use of
underutilized resources, we have reported the
preparation and characterization of collagens
from aquatic organisms, mainly marine
verte-brates and inverteverte-brates (Nagai et al., 1999, 2000,
2001, 2002; Nagai & Suzuki, 2000a, b, c, 2002a, b)
Although there are many reports about collagen
from skin of marine organisms, there are few
studies of fish scales except for the studies of
Kimura’s group (Kimura et al., 1991) and those of
Shirai (Nomura et al., 1996) Kimura et al (1991)
reported that collagen from carp scale could be
extracted with 0.5 m acetic acid and the yield was
about 7% on dry weight basis On the contrary, Nomura et al (1996) reported the extraction of collagen from sardine scale with different solvent systems: 0.05 m Tris–HCl (pH 7.5) containing 0.5 m ethylenediaminetetraacetic acid (EDTA) Its yield was very low, about 5% It is possible for fish scales to have potential as an important source of collagen because they contain a large quantity of collagen This paper describes the preparation and characterization of collagen from fish scales
Materials and methods Fish
Fish sardine Sardinops melanostictus (body weight 0.1–0.2 kg), red sea bream Pagrus major (1.0–
1.3 kg) and Japanese sea bass Lateolabrax japon-icus (0.8–1.2 kg) were purchased from a fish market in Shimonoseki City, Yamaguchi Prefec-ture, Japan The scales were removed, washed with distilled water and lyophilized
Preparation of scale collagen All the preparative procedures were at 4C The lyophilized scales (5.0 g) were treated with 0.1 n NaOH to remove noncollagenous proteins and
*Correspondent: Fax: +81 832 33 1816;
e-mail: machin@fish-u.ac.jp
Trang 2pigments for 3 days by changing the solution once a
day, then washed with distilled water, dried, and
stored at )85 C until used The matter was
extracted with 0.5 m acetic acid for 3 days, and
the extract was centrifuged at 50 000 g for 1 h The
supernatants were pooled and salted out by adding
NaCl to a final concentration of 0.9 m
Unfortu-nately, the collagen was not precipitated in this
solution The resultant matter, obtained by
centrif-ugation at 50 000 g for 1 h, was decalcified with
0.05 m Tris–HCl (pH 7.5) containing 0.5 m
EDTA-4 Na for 2 days and then disaggregated with 0.1 m
Tris–HCl (pH 8.0) containing 0.5 m NaCl, 0.05 m
EDTA-2 Na and 0.2 m 2-mercaptoethanol (2-ME)
for 3 days After collecting the collagen fibrils with
cheesecloth, the residue was washed with distilled
water for 2 days by changing the water once a day
The residue obtained was lyophilized The
lyoph-ilized fibrils were suspended in 0.5 m acetic acid and
digested with 10% (w/w) pepsin (EC 3.4.23.1; 2·
crystallized, 3085 U mg)1protein; Sigma, USA) at
4C for 24 h The pepsin-solubilized collagen was
centrifuged at 50 000 g for 1 h and the supernatant
dialyzed against 0.02 m Na2HPO4 (pH 7.2) for
3 days, changing the solution once a day The
resultant precipitate, obtained by centrifugation at
50 000 g for 1 h, was dissolved in 0.5 m acetic acid
and was salted out by adding NaCl to a final
concentration of 0.9 m, followed by precipitation of
the collagen by the addition of a final concentration
of 2.4 m NaCl at neutral pH The resultant
preci-pitate was obtained by centrifugation at 50 000 g
for 1 h, dissolved in 0.5 m acetic acid, and then
lyophilized
Sodium dodecyl sulphate-polyacrylamide gel
electrophoresis
Sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE) was performed as
described previously (Nagai et al., 2002) After
the electrophoresis, the gels were stained with
Coomassie Brilliant Blue R-250 (Fluka Fine
Chemical Co Ltd., Tokyo, Japan) and destained
with 5% methanol and 7.5% acetic acid
Peptide mapping
The collagen samples (0.5 mg) were dissolved in
0.1 m sodium phosphate buffer (pH 7.2)
contain-ing 0.5% SDS and heated at 100C for 5 min After cooling in ice, the digestion was done at
37C for 30 min using 5 lL of lysyl endopepti-dase from Achromobacter lyticus (EC 3.4.21.50; 4.5 amidase activity mg)1 protein; Wako Pure Chemicals, Osaka, Japan) After adding SDS to a final concentration of 2%, the proteolysis was stopped by boiling for 5 min SDS-PAGE was performed by the method of Laemmli (1970) using 15% gels
Subunit composition
To separate the subunits of each collagen sample, the sample was applied to a CM-Toyopearl 650M (Tosoh Co., Tokyo, Japan) column chromatog-raphy Fifteen milligrams of the collagen sample were dissolved in 20 mm sodium acetate buffer (pH 4.8) containing 6 m urea at 4C, denatured
at 45C for 30 min, and the solution was centrifuged at 50 000 g at 20C for 1 h The supernatants were applied to a CM-Toyopearl 650M column (1.0· 6.0 cm) previously equili-brated with the same buffer Each subunit was eluted with a linear gradient of 0–0.15 m NaCl in the same buffer at a flow rate of 0.8 mL min)1 The subunit quantity was detected by using absorbance at 230 nm, and the fractions were examined by SDS-PAGE
Denaturation temperature Denaturation temperature (Td) was measured by the method of Nagai et al (2002) Five millilitres
of a 0.03% collagen solution in 0.1 m acetic acid was used for viscosity measurements Tdwas the temperature where the change in viscosity using a Canon–Fenske type viscometer with an average shear gradient of 400 s)1, was half completed
Amino acid composition Collagen samples were hydrolyzed under reduced pressure in 6 m HCl at 110C for 24 h, and the hydrolysates were analysed on a JASCO liquid-chromatography system by on-line precolumn derivatization with OPA This system consisted
of a JASCO PU-2080 plus intelligent HPLC-pump, a JASCO FP-2020 plus intelligent fluores-cence detector, a JASCO CO-2060 plus intelligent
Trang 3column thermostat, a JASCO DG-2083-53 3-line
degasser, a JASCO LG-2080-02 ternary gradient
unit, a JASCO AS-2057 plus intelligent sampler,
and a JASCO CrestPak C18S (/ 4.6· 150 mm)
reversed-phase column The excitation and
emis-sion wavelengths were set at 345 and 455 nm,
respectively Eluents were filtered through
Milli-pore membrane filters (Milli-pore size 0.45 lm)
Results and discussion
The scales were hardly solubilized with 0.5 m
acetic acid The supernatants obtained by
centri-fugation were salted out by adding NaCl
Unfor-tunately the collagen was not precipitated in the
sample solution As a result of decalcification and
disaggregation procedures, the collagen was easily
solubilized by limited pepsin proteolysis
Colla-gens solubilized by pepsin were effectively purified
by differential salt precipitation The yields of the
collagens were very high and were in the range of
about 38–51% on a dry weight basis (sardine
50.9%, red sea bream 37.5% and Japanese sea
bass 41.0%, respectively) The results were similar
to previous reports (Nagai et al., 1999, 2000, 2001,
2002; Nagai & Suzuki, 2000a,b,c, 2002a,b),
suggesting that a great amount of collagen can
be obtained from aquatic animals However,
Nomura et al (1996) prepared collagen from
sar-dine scale with different solvent systems: 0.05 m
Tris-HCl (pH 7.5) containing 0.5 m EDTA
Fur-thermore, they reported that the yield of the
collagen was only 5%, as acid solubilized collagen
The preparative method reported here in was
superior to earlier reports and the collagen was
recovered in high yield from fish scale The
colla-gens obtained were examined by SDS-PAGE using
3.5% gel It was found that the collagens from red
sea bream and Japanese sea bass comprised only
one a chain, a1, although red sea bream collagen
seemed to have a3 chain (Fig 1) On the contrary,
sardine collagen had at least two different a chains,
a1 and a2 (Fig 1) The a chains of these collagens
were different when compared with those from
porcine skin a chains It suggests that these
colla-gens are different to one another in primary
structure In this electrophoretic separation the a3
chain was not separated from the corresponding a1
chain if other a chains, such as a3 and a4, were
present in these scale collagens
To compare the patterns of peptide fragments with fish scale and porcine collagens, the digested collagens were applied to SDS-PAGE using 15%
Figure 1 Sodium dodecyl sulphate-polyacrylamide gel elec-trophoresis of porcine skin type I collagen and fish scale collagens on 3.5% gels containing 3.5 m urea (a) Porcine, (b) sardine, (c) red sea bream and (d) Japanese sea bass.
Figure 2 Peptide mapping of lysyl endopeptidase digests from several fish scale collagens (a) High molecular marker, (b) porcine, (c) sardine, (d) Japanese sea bass, (e) red sea bream and (f) low molecular marker.
Trang 4gel The electrophoretic patterns of the three fish
scale collagens were similar to each other (Fig 2)
In particular the protein bands with molecular
mass of 200, 120 or 30–40 kDa were nearly
identical in all these fish species The pattern of
peptide fragments of porcine skin collagen was
quite different from those of other fish scale
collagens, although the pattern of porcine collagen
also shows some similarities in comparison with
those of fish scale collagens (Fig 2)
The denatured collagens were resolved by CM-Toyopearl 650M column chromatography to determine the subunit composition of fish scale collagens The chromatographic fractions were identified by SDS-PAGE and sardine collagen showed two a chains; a1 and a2 (Fig 3) Simi-larly, red sea bream (Fig 4) and Japanese sea bass (Fig 5) collagens comprised two a chains Although a band corresponding to a3 in Japanese sea bass collagen was detected, it seemed to be partially denatured The scale collagens were heterotrimers with a chain composition of (a1)2a2 Kimura et al (1991) prepared collagen from carp scale and reported the properties Carp
Figure 3 CM-Toyoperal 650M column chromatography of
denatured sardine scale collagen A 1.0 · 5.0 column of
CM-Toyopearl 650M was equilibrated with 0.02 m sodium
acetate buffer (pH 4.8) containing 6 m urea, and maintained
at 37 C The collagen sample (15.0 mg) was dissolved in
5 mL of the same buffer, denatured for 30 min at 45 C, and
then eluted from the column with a linear gradient of 0 to
0.15 m NaCl at a flow rate of 0.8 mL min)1 The fractions
indicated by the numbers were examined by sodium dodecyl
sulphate-polyacrylamide gel electrophoresis.
Figure 4 CM-Toyoperal 650M column chromatography of denatured red sea bream scale collagen The chromato-graphic conditions are shown in Fig 3.
Trang 5scale collagen had three different a chains; a1, a2
and a3, giving a heterotrimer with a chain
composition of a1a2a3
To determine the Td of the scale collagens
separated in three experiments, the changes in
viscosity and the Tdwere calculated from thermal
denaturation curves It was calculated that the
Tds of fish scale collagens were as follows: sardine
28.5C, red sea bream 28.0 C and Japanese sea
bass 28.0C (Fig 6) On the contrary, the Tdof
porcine skin collagen was measured at 37.0C,
this is about 9C higher than those of fish scale It
was suggested that the tendency for the Td of
marine organism to be lower than that of land
animals is correlated with their environmental and
body temperature (Rigby, 1968)
The amino acid composition in three fish scale collagens is shown as residues per 1000 total residues (Table 1) Glycine was the most abundant amino acid in all of these collagens and the value
Figure 5 CM-Toyoperal 650M column chromatography of
denatured Japanese sea bass scale collagen The
chromato-graphic conditions are shown in Fig 3.
Figure 6 Thermal denaturation curve of fish scale collagen solutions as measured by viscosity in 0.1 m acetic acid The incubation time at each temperature was 30 min Collagen concentration: 0.03%; (s) porcine skin collagen, (d) sardine collagen, (h) red sea bream collagen, (+) Japanese sea bass collagen.
Table 1 Amino acid composition of scale collagens from fish species, residues/1000
Amino acid Sardine Red sea bream Japanese sea bass Hydroxyproline 86 87 85
Aspartic acid 47 46 48 Threonine 24 26 25 Serine 41 39 42 Glutamic acid 71 72 75 Proline 111 109 108 Glycine 340 340 341 Alanine 115 116 114 Half-cystine 2 2 2 Valine 18 19 18 Methionine 13 12 12 Isoleucine 11 10 10 Leucine 22 22 23 Tyrosine 3 2 2 Phenylalanine 12 13 13 Tryptophan 0 0 0 Lysine 25 23 24 Histidine 7 7 6 Arginine 52 55 52 Total 1000 1000 1000
Trang 6was approximately 340/1000 residues Alanine,
proline, hydroxyproline and glutamic acid had
relatively high contents in these collagens On the
contrary, tryptophan was not detected in any
collagen samples
It is known that the major components in fish
scale are as follows: water 70%, protein 27%, lipid
1% and ash 2% Organic compounds comprise
40–90% in scales and most of them are collagen,
regardless of fish species At present, great
quan-tities of fish scales are produced in fish shops and
fish-processing factories However, the effective
use of these scales is minimal In this study,
collagen obtained from three types of fish scales
possessed properties typical of type I collagen
Among them, surprisingly, sardine scale showed
the highest yield of collagen, about 51.0% on a dry
weight basis From these results it is clear that fish
scales have the potential to be an alternative
source of collagen to porcine and cattle skin and
bone Unless the problem of BSE infection in land
animals is resolved, fish scale as an alternative
source of collagen, will attract much attention in
the cosmetic and medical fields
Acknowledgments
This work was supported in part by the grant from
the Kiei-Kai Research Foundation, Tokyo, Japan
We would like to express our heartfelt gratitude to
the donor
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