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.
International Journal of Food Science and Technology 2004, 39, 239–244 Fish scale collagen Preparation and partial characterization Takeshi Nagai,1* Masami Izumi2 & Masahide Ishii3 Department of Food Science and Technology, National Fisheries University, Shimonoseki, Yamaguchi 7596595, Japan Ribro Com, Inc., 1-5-10 Nishi-shinbashi, Minato-ku, Tokyo 1050003, Japan 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 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 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 vertebrates and invertebrates (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 *Correspondent: Fax: +81 832 33 1816; e-mail: machin@fish-u.ac.jp doi:10.1111/j.1365-2621.2004.00777.x Ó 2004 Blackwell Publishing Ltd 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 japonicus (0.8–1.2 kg) were purchased from a fish market in Shimonoseki City, Yamaguchi Prefecture, Japan The scales were removed, washed with distilled water and lyophilized Preparation of scale collagen All the preparative procedures were at °C The lyophilized scales (5.0 g) were treated with 0.1 n NaOH to remove noncollagenous proteins and 239 240 Fish scale collagen T Nagai et al pigments for 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 days, and the extract was centrifuged at 50 000 g for h The supernatants were pooled and salted out by adding NaCl to a final concentration of 0.9 m Unfortunately, the collagen was not precipitated in this solution The resultant matter, obtained by centrifugation at 50 000 g for h, was decalcified with 0.05 m Tris–HCl (pH 7.5) containing 0.5 m EDTA4 Na for 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 days After collecting the collagen fibrils with cheesecloth, the residue was washed with distilled water for days by changing the water once a day The residue obtained was lyophilized The lyophilized 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)1 protein; Sigma, USA) at °C for 24 h The pepsin-solubilized collagen was centrifuged at 50 000 g for h and the supernatant dialyzed against 0.02 m Na2HPO4 (pH 7.2) for days, changing the solution once a day The resultant precipitate, obtained by centrifugation at 50 000 g for 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 precipitate was obtained by centrifugation at 50 000 g for 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 100 °C for After cooling in ice, the digestion was done at 37 °C for 30 using lL of lysyl endopeptidase 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 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 chromatography Fifteen milligrams of the collagen sample were dissolved in 20 mm sodium acetate buffer (pH 4.8) containing m urea at °C, denatured at 45 °C for 30 min, and the solution was centrifuged at 50 000 g at 20 °C for h The supernatants were applied to a CM-Toyopearl 650M column (1.0 · 6.0 cm) previously equilibrated 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 Td was 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 m HCl at 110 °C for 24 h, and the hydrolysates were analysed on a JASCO liquidchromatography system by on-line precolumn derivatization with OPA This system consisted of a JASCO PU-2080 plus intelligent HPLCpump, a JASCO FP-2020 plus intelligent fluorescence detector, a JASCO CO-2060 plus intelligent International Journal of Food Science and Technology 2004, 39, 239–244 Ó 2004 Blackwell Publishing Ltd Fish scale collagen T Nagai et al column 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 emission wavelengths were set at 345 and 455 nm, respectively Eluents were filtered through Millipore membrane filters (pore size 0.45 lm) Results and discussion The scales were hardly solubilized with 0.5 m acetic acid The supernatants obtained by centrifugation were salted out by adding NaCl Unfortunately 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 Collagens 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 sardine scale with different solvent systems: 0.05 m Tris-HCl (pH 7.5) containing 0.5 m EDTA Furthermore, 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 collagens 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 collagens 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 Ó 2004 Blackwell Publishing Ltd a b c d Figure Sodium dodecyl sulphate-polyacrylamide gel electrophoresis 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 To compare the patterns of peptide fragments with fish scale and porcine collagens, the digested collagens were applied to SDS-PAGE using 15% a b c d e f Figure 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 International Journal of Food Science and Technology 2004, 39, 239–244 241 242 Fish scale collagen T Nagai et al gel 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) Figure 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 m urea, and maintained at 37 °C The collagen sample (15.0 mg) was dissolved in mL of the same buffer, denatured for 30 at 45 °C, and then eluted from the column with a linear gradient of 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 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) Similarly, 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 CM-Toyoperal 650M column chromatography of denatured red sea bream scale collagen The chromatographic conditions are shown in Fig International Journal of Food Science and Technology 2004, 39, 239–244 Ó 2004 Blackwell Publishing Ltd Fish scale collagen T Nagai et al Figure 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 Collagen concentration: 0.03%; (s) porcine skin collagen, (d) sardine collagen, (h) red sea bream collagen, (+) Japanese sea bass collagen Table Amino acid composition of scale collagens from fish species, residues/1000 Amino acid Figure CM-Toyoperal 650M column chromatography of denatured Japanese sea bass scale collagen The chromatographic conditions are shown in Fig scale 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 Td were calculated from thermal denaturation curves It was calculated that the Td s of fish scale collagens were as follows: sardine 28.5 °C, red sea bream 28.0 °C and Japanese sea bass 28.0 °C (Fig 6) On the contrary, the Td of porcine skin collagen was measured at 37.0 °C, this is about °C 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) Ó 2004 Blackwell Publishing Ltd Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Lysine Histidine Arginine Total Sardine Red sea bream Japanese sea bass 86 47 24 41 71 111 340 115 18 13 11 22 12 25 52 87 46 26 39 72 109 340 116 19 12 10 22 13 23 55 85 48 25 42 75 108 341 114 18 12 10 23 13 24 52 1000 1000 1000 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 International Journal of Food Science and Technology 2004, 39, 239–244 243 244 Fish scale collagen T Nagai et al was 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 quantities 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 References Kimura, S., Miyauchi, Y & Uchida, N (1991) Scale and bone type I collagens of carp (Cyprinus carpio) Comparative Biochemistry and Physiology, 99B, 473–476 Laemmli, U.K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature, 227, 680–685 Nagai, T & Suzuki, N (2002a) Collagen of the skin of ocellate puffer fish (Takifugu rubripes) Food Chemistry, 78, 173–177 Nagai, T., Nagamori, K., Yamashita, E & Suzuki, N (2002) Collagen of octopus Callistoctopus arakawai arm International Journal of Food Science & Technology, 37, 285–289 Nagai, T., Ogawa, T., Nakamura, T et al (1999) Collagen of edible jellyfish exumbrella Journal of the Science of Food and Agriculture, 79, 855–858 Nagai, T & Suzuki, N (2000a) Isolation of collagen from fish waste material-skin, bone and fins Food Chemistry, 68, 277–281 Nagai, T & Suzuki, N (2000b) Partial characterization of collagen from purple sea urchin (Anthocidaris crassispina) test International Journal of Food Science & Technology, 35, 497–501 Nagai, T & Suzuki, N (2000c) Preparation and characterization of several fish bone collagens Journal of Food Biochemistry, 24, 427–436 Nagai, T & Suzuki, N (2002b) Preparation and partial characterization of collagen from paper nautilus (Argonauta argo, Linnaeus) outer skin Food Chemistry, 76, 149–153 Nagai, T., Worawattanamateekul, W., Suzuki, N et al (2000) Isolation and characterization of colagen from rhizostomous jellyfish (Rhopilema asamushi) Food Chemistry, 70, 205–208 Nagai, T., Yamashita, E., Taniguchi, K., Kanamori, N & Suzuki, N (2001) Isolation and characterisation of collagen from the outer skin waste material of cuttlefish (Sepia lycidas) Food Chemsitry, 72, 425–429 Nomura, Y., Sakai, H., Ishii, Y & Shirai, K (1996) Preparation and some properties of type I collagen from fish scales Bioscience, Biotechnology, and Biochemistry, 60, 2092–2094 Rigby, B.J (1968) Amino-acid composition and thermal stability of the skin collagen of the Antarctic ice-fish Nature, 219, 166–167 Yamauchi, K (2002) Bovine Spongiform Encephalopathy and People Tokyo, Japan: Iwanami Press International Journal of Food Science and Technology 2004, 39, 239–244 Ó 2004 Blackwell Publishing Ltd