Food Chemistry 78 (2002) 173–177

www.elsevier.com/locate/foodchem

Collagen of the skin of ocellate puffer fish

(Takifugu rubripes)
Takeshi Nagaia,*, Yoko Arakib, Nobutaka Suzukia
a
Department of Food Science and Technology, National Fisheries University, Shimonoseki, Yamaguchi 7596595, Japan
b
Faculty of Home Economics, Tokyo Kasei Gakuin University, Machida, Tokyo 1940292, Japan

Received 20 September 2001; received in revised form 21 November 2001; accepted 21 November 2001

Abstract
Collagens (acid-solubilized and pepsin-solubilized collagens) were prepared from ocellate puffer fish skin and partially char-
acterized. With respect to the pepsin-solubilized collagen, it was a heterotrimer with a chain composition of (a1)2a2. The patterns of
peptide fragments were different from skin collagens of other species. The denaturation temperature was 28  C, about 9  C lower
than that of porcine skin collagen. On the other hand, the yields of acid-solubilized and pepsin-solubilized collagens were very
high, 10.7% and 44.7%, respectively, on a dry weight basis. These results suggest that ocellate puffer fish skin has potential as an
alternative source of collagen for use in various fields. # 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Collagen; Ocellate puffer fish; Yield; Underutilized resources

1. Introduction Collagen is the predominant protein in the living

body. The main sources of industrial collagen are limited
Edible puffer fish, such as Takifugu rubripes, Takifugu to those from pig and bovine skin and bones. It is
porphyreus, and Takifugu vermicularis, belong to the known that the skin of puffer fish contains a large
order Tetraodontidae. It is known that the ovary and quantity of collagen, and that skin of T. rubripes has
liver of many puffer fish contain a toxin, tetrodotoxin, potential as an important source of collagen. In this
one of the most potent non-protein neurotoxins. Puff- paper, the preparation and characterisation of collagen
erfish of the genus Takifugu are mainly distributed in from the skin of T. rubripes are described.
the East China Sea and its surrounding waters, in which
about 23 species have been recognized (Masuda, Taka-
hashi, Dotsu, Miyaki, Tabeta, & Matsuura, 1986). 2. Materials and methods
Among them, T. rubripes is treated as a fish of the
highest quality in Japan. It is covered with thick skin, 2.1. Fish
and its skin is non-toxic. The Japanese eat the skin as a
garnishing of sliced raw fish, sashimi, and as a vinegared Ocellate puffer fish, T. rubripes, were purchased from
dish. Moreover, the Japanese eat its muscle as sashimi, a the local wholesale market in Shimonoseki City, Yama-
pot of fish and vegetables cooked before the dinners guchi Prefecture, Japan. The skins were removed, cut
chiri-nabe, and miso soup (Japan Association of Training into small pieces, and stored at 25  C until required.
Colleges for Cooks, 1996).
2.2. Preparation of skin collagen

The collagen was prepared by the method of Nagai

and Suzuki (2000a). All the preparative procedures were
* Corresponding author. Tel.: +81-832-86-5111; ext. 407; fax:
performed at 4  C. The skins were treated with 0.1 N
+81-832-33-1816. NaOH to remove noncollagenous proteins and pigments,
E-mail address: machin@fish-u.ac.jp (T. Nagai). then washed with distilled water, and lyophilized. To
0308-8146/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0308-8146(01)00396-X
174 T. Nagai et al. / Food Chemistry 78 (2002) 173–177

remove fat in the lyophilized powder, it was treated 2.5. Peptide mapping
with 10% butyl alcohol for 2 days once per day,
washed with distilled water, and lyophilized. The matter The collagen sample was digested by lysyl endopepti-
was extracted with 0.5 M acetic acid for 3 days, and dase from Achromobacter lyticus (EC 3. 4. 21. 50; 4.5
the extract was centrifuged at 20,000g for 1 h. The amidase activity/mg protein; Wako Pure Chemicals,
supernatants were salted-out by adding NaCl to a final Osaka, Japan), applied to 15% gel. SDS-PAGE was
concentration of 0.7 M and followed by precipitation performed by the method of Laemmli (1970).
of the collagen by the addition of NaCl (final con-
centration of 2.3 M) in 0.05 M Tris–HCl (pH 7.5). The 2.6. Denaturation temperature
resultant precipitate was separated by centrifugation at
20,000g for 1 h, and dissolved in 0.5 M acetic acid. The denaturation temperature (Td) was measured by
The solution obtained was dialyzed against 0.1 M acetic the method of Nagai et al. (2000). Td was determined as
acid solution, distilled water, and then lyophilized (acid- the temperature at which the change in viscosity, using a
solubilized collagen; ASC). The residue was suspended Canon-Fenske type viscometer with an average shear
in 0.5 M acetic acid and digested with 10% (w/v) pepsin gradient of 400 s 1, was half completed.
(EC 3. 4. 23. 1; 2crystallized; 3085 U/mg protein,
Sigma, USA) for 48 h at 4  C. The viscous solution was 2.7. Amino acid composition
centrifuged at 20,000g for 1 h and the supernatants
were dialyzed against 0.02 M Na2HPO4 (pH 7.2) for 3 A collagen sample was hydrolyzed under reduced
days with a change of solution once per day. After the pressure in 6 M HCl at 110 C for 24 h, and the hydro-
dialysate was centrifuged at 20,000g for 1 h, the pre- lysates were analyzed on a Shimadzu amino acid analy-
cipitate was dissolved in 0.5 M acetic acid and was sal- zer (LC-10A).
ted-out by adding NaCl to a final concentration of 0.7
M and followed by precipitation by addition of NaCl
(final concentration of 2.2 M) in 0.05 M Tris–HCl (pH 3. Results and discussion
7.5). The resultant precipitate was separated by cen-
trifugation at 20,000g for 1 h, dissolved in 0.5 M The skin of ocellate puffer fish was not completely
acetic acid, dialyzed against the same solution, and solubilized with 0.5 M acetic acid. In this way it was simi-
then lyophilized (pepsin-solubilized collagen; PSC). lar to the skin of Callistoctopus arakawai arm (Nagai,
Nagamori, Yamashita, & Suzuki, 2001), cuttlefish outer
2.3. SDS-polyacrylamide gel electrophoresis (SDS- skin (Nagai, Yamashita, Taniguchi, Kanamori, & Suzuki,
PAGE) 2001) and paper nautilus outer skin (Nagai & Suzuki,
2001). This result was different from those for Japanese
SDS-PAGE was performed as previously described sea bass, chub mackerel, and bullhead shark skin (Nagai
(Nagai & Suzuki, 2000c). After electrophoresis, each & Suzuki, 2000a). The collagen of ocellate puffer fish skin
gel was stained with Coomassie Brilliant Blue R-250 was easiliy solubilized by limited pepsin proteolysis. This
(Fluka Fine Chemical Co., Ltd., Tokyo, Japan) and was similar to those of edible jellyfish exumbrella (Nagai
destained with 5% methanol and 7.5% acetic acid. et al., 1999) and rhizostomous jellyfish mesogloea (Nagai
et al., 2000). PSC was effectively purified by differential
2.4. Subunit composition salt precipitation. The yield of PSC was very high (44.7%
on a dry weight basis). On the other hand, the yield of
To separate the subunit components of this col- ASC was low (10.7% on a dry weight basis). This result
lagen, the collagen sample was applied to a CM- was similar to those for fish skin (Japanese sea bass,
Toyopearl 650M (Tosoh Co. Tokyo, Japan) column. 51.4%, chub mackerel, 49.8%, and bullhead shark,
Briefly, 20 mg of the collagen sample were dissolved 50.1%) (Nagai & Suzuki, 2000a), purple sea urchin test
in 20 mM sodium acetate buffer (pH 4.8) containing (35.0%) (Nagai & Suzuki, 2000b), fish bone (Japanese sea
6 M urea at 4  C, denatured at 45  C for 30 min, bass, 40.7%, horse mackerel, 43.5%, and ayu, 53.6%)
and the solution centrifuged at 20,000g at 20  C for 1 (Nagai & Suzuki, 2000c), edible jellyfish exumbrella
h. The supernatants were applied to a CM-Toyopearl (46.4%) (Nagai et al., 1999), rhizostomous jellyfish
650M column (1.05.0 cm), previously equilibrated mesogloea (35.2%) (Nagai et al., 2000), C. arakawai
with the same buffer. Each subunit was eluted with a arm (62.9%) (Nagai, Nagamori, Yamashita, & Suzuki
linear gradient of 0–0.15 M NaCl in the same buffer at et al., 2001), cuttlefish outer skin (35.0%) (Nagai,
a flow rate of 0.8 ml/min. The subunit components Yamashita, Taniguchi, Kanamori, & Suzuki, 2001),
were detected by absorbance at 230 nm. The fractions and paper nautilus outer skin (50.0%) (Nagai & Suzuki,
indicated by the numbers were examined by SDS- 2001). It appears that a large amount of collagen can be
PAGE. obtained from aquatic animals. The collagen obtained was
 T. Nagai et al. / Food Chemistry 78 (2002) 173–177 175

examined by SDS-PAGE using 3.5% gel. It was found The collagens digested by lysyl endopeptidase were
that these collagens comprised at least two different a applied to 15% gel SDS-PAGE to compare the patterns
chains;a 1 and a 2 (Fig. 1). In electrophoretic mobility, the of peptide fragment with porcine and other fish species
positions of a chains of these collagens differed from those skin collagens. As a result, the electrophoretic pattern of
of porcine skin a chains. That is, a chains of ocellate puffer ocellate puffer PSC was similar to that of ASC (Fig. 3).
fish skin collagen are distinct in this primary structure. If
other a chains, such as a3 and a4, were present in these
collagens, they were not separated from the corresponding
a 1 chain under the electrophoretic conditions employed.
To determine the subunit composition of ocellate
puffer fish skin PSC, the denatured collagen was further
resolved by CM-Toyopearl 650M column chroma-
tography and its chromatographic fractions were identi-
fied by SDS-PAGE. It was shown that PSC consists of
two a chains (Fig. 2). This collagen is a heterotrimer
with a chain composition of (a1)2a2. Kimura, Ohno,
Miyauchi, and Uchida (1987) examined the fish skin
collagens and reported that the a 3 chain was widely
distributed in teleosts, such as eel, sardine, chum sal-
mon, rainbow trout, carp, anger, Alaska pollack, cod,
halfbeak, common mackerel, tilapia, red barracuda,
northern dab, and file fish. In the previous paper
(Kimura, 1985; Kimura & Ohno, 1987; Kimura et al.,
1987; Piez, 1965), it was reported that the a 3 chain was
detected in 14 fish species of 17 teleosts. Moreover, we
have reported the existence of a 3 chain in edible jellyfish
exumbrella (Nagai et al., 1999), rhizostomous jellyfish
mesogloea (Nagai et al., 2000), ayu bone (Nagai &
Suzuki, 2000c), paper nautilus outer skin (Nagai &
Suzuki, 2001), and C. arakawai arm (Nagai, Nagamori,
Yamashita, & Suzuki, 2001a). Now our present data are
different from those of previous papers.

Fig. 2. CM-Toyopearl 650M column chromatography of denatured

ocellate puffer fish skin collagens. The fractions indicated by the
numbers were examined by SDS-PAGE.

Fig. 3. Comparison, by peptide mapping, of lysyl endopeptidase digests

Fig. 1. SDS-polyacrylamide gel electrophoresis of porcine skin type I from several fish skin collagens. (A) Porcine collagen; (B) ocellate puffer
collagen and ocellate puffer fish skin collagens on 3.5% gels containing fish acid-solubilized collagen; (C) ocellate puffer fish pepsin-solubilized
3.5 M urea. (A) Porcine; (B) ocellate puffer fish acid-solubilized col- collagen; (D) chub mackerel collagen; (E) Japanese sea bass collagen; (F)
lagen; (C) ocellate puffer fish pepsin-solubilized collagen. yellowtail collagen; (G) bullhead shark collagen; and (H) ayu collagen.
176 T. Nagai et al. / Food Chemistry 78 (2002) 173–177

Table 1
Amino acid composition of ocellate puffer fish skin pepsin-solubilized
collagen, residues/1000

Amino acid

Hydroxyproline 67
Aspartic acid 50
Threonine 25
Serine 48
Glutamic acid 87
Proline 103
Glycine 351
Alanine 106
Half-cystine 2
Valine 17
Methionine 14
Isoleucine 12
Leucine 23
Tyrosine 4
Phenylalanine 10
Tryptophan 0
Fig. 4. Thermal denaturation curve of ocellate puffer fish skin col-
Lysine 19
lagen solution as measured by viscosity in 0.1 M acetic acid. The
Histidine 8
incubation time at each temperature was 30 min. Collagen concentra-
Arginine 54
tion: 0.03%; (*): porcine skin collagen; (*): ocellate puffer fish skin
collagen.
Total 1000

Although the patterns of high molecular weight fragments those of land animals is correlated with their environ-
were similar to each other, the patterns of these collagens mental and body temperatures (Rigby, 1968).
were different from other species. It appears that the pri- The amino acid composition, expressed as residues
mary structure of collagen differs among species of teleosts. per 1000 total residues, is shown in Table 1. This shows
The Td of ocellate puffer PSC was calculated from the that glycine was the most abundant amino acid in ocel-
thermal denaturation curve. For comparison, Td of por- late puffer skin collagen and that there were relatively
cine skin collagen was similarly measured. It was calcu- high contents of alanine, proline and glutamic acid,
lated that Td of ocellate puffer PSC was about 28  C decreasing in that order. Glycine accounted for more
(Fig. 4). This was about 9  C lower than that of porcine than 30% of all amino acids in this collagen. Its value
skin collagen. This value was similar to those obtained was approximately 351 residues. The degree of hydro-
from other marine organisms: Alaska pollack skin xylation of proline was calculated to be 39.4%. In our
(16.8  C) and swim bladder (18.4  C) (Kimura & Ohno, previous paper, it was reported that the degrees of
1987), muscles of carp (32.5  C), eel (30.2  C), common hydroxylation of proline were as follows: 32.8% (edible
mackerel (26.9  C), saury (24.0  C), chum salmon jellyfish exumbrella: Nagai et al., 1999), 48.0% (purple
(20.6  C) and skins of carp (31.7  C), eel (29.3  C), sea urchin test: Nagai & Suzuki, 2000b), 51.7% (C.
common mackerel (26.1  C), saury (23.0  C), chum sal- arakawai arm: Nagai, Nagamori, Yamashita, & Suzuki,
mon (19.4  C) (Kimura, Zhu, Matsui, Shijoh, & Taka- 2001a), and 47.9% (cuttlefish outer skin: Nagai, Yama-
mizawa, 1988); body wall of starfish (23.0  C) (Kimura, shita, Taniguchi, Kanamori, & Suzuki, 2001). Among
Omura, Ishida, & Shirai, 1993), edible jellyfish exum- them, it was found that ocellate puffer skin collagen was
brella (26.0  C) (Nagai et al., 1999); skin of Japanese sea unstable against temperature.
bass (26.5  C), chub mackerel (25.6  C), bullhead shark In conclusion, a great quantity of collagen could be
(25.0  C), bone of Japanese sea bass (30.0  C), skipjack obtained from ocellate puffer skin. This pufferfish is the
and ayu (29.7  C), yellow sea bream and horse mackerel bigest among about 23 puffer species that have been
(29.5  C) and Japanese sea bass fin (29.1  C) (Nagai & recognized in the East China Sea and its surrounding
Suzuki, 2000a), rhizostomous jellyfish mesogloea (28.8  C) waters, and its skin is non-toxic like those of T. rubripes,
(Nagai et al., 2000), purple sea urchin test (28.0  C) (Nagai T. xanthopterus, Lagocephalus laevigatus inermis, Lago-
& Suzuki, 2000b), C. arakawai arm (28.0  C) (Nagai, cephalus wheeleri, and Liosaccus pachygaster. For these
Nagamori, Yamashita, & Suzuki, 2001a), (cuttlefish outer reasons, ocellate puffer skin has potential as an alter-
skin (27.0  C) (Nagai, Yamashita, Taniguchi, Kanamori, native source of collagen to cattle and porcine skin and
& Suzuki, 2001b); and paper nautilus outer skin (27.0  C) bone. This study is only one useful report of many
(Nagai & Suzuki, 2001). It has been suggested that the studies into making more effective use of underutilized
tendency for Tds of marine organism to be lower than resources such as collagen.
 T. Nagai et al. / Food Chemistry 78 (2002) 173–177 177

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the Kiei-Kai Research Foundation, Tokyo, Japan. We lagen of octopus Callistoctopus arakawai arm. International Journal
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K., Nakao, M., & Yano, T. (1999). Collagen of edible jellyfish
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