Food

Chemistry
Food Chemistry 93 (2005) 475–484
www.elsevier.com/locate/foodchem

Isolation and characterisation of acid and pepsin-solubilised

collagens from the skin of Brownstripe red snapper (Lutjanus vitta)
Akkasit Jongjareonrak a, Soottawat Benjakul a,*, Wonnop Visessanguan b,
Takeshi Nagai c, Munehiko Tanaka d
a
Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
b
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency,
113 Paholyothin Rd., Klong1, Klong Luang, Pathumthani 12120, Thailand
c
Department of Food Science and Technology, Tokyo University of Agriculture, Hokkaido 0991493, Japan
d
Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Konan 4, Minato, Tokyo 108-8477, Japan

Received 12 July 2004; received in revised form 4 October 2004; accepted 4 October 2004

Abstract

Acid-solubilised collagen (ASC) and pepsin-solubilised collagen (PSC) were successfully extracted from the skin of Brownstripe
red snapper (Lutjanus vitta) with yields of 9% and 4.7%, respectively, on the basis of wet weight. Both ASC and PSC consisted of two
different a chains (a1 and a2), and were characterised to be type I with no disulfide bond. PSC had a lower content of high molecular
weight cross-links, than did ASC. Peptide maps of ASC and PSC hydrolysed by V8 protease and lysyl endopeptidase showed some
differences in peptide patterns between the two fractions and were totally different from those of calf skin collagen type I, suggesting
differences in amino acid sequences and collagen conformation. Maximum solubility in 0.5 M acetic acid was observed at pH 3 and
pH 4 for ASC and PSC, respectively. A sharp decrease in solubility was observed in the presence of NaCl, above 2% and 3%, (w/v)
for ASC and PSC, respectively. Tmax values of both collagen fractions were similar and shifted to a lower value in the presence of
acetic acid, suggesting some changes in the collagen structure caused by acid induction.

2004 Published by Elsevier Ltd.

Keywords: Collagen; Brownstripe red snapper; Skin; ASC; PSC; Solubility

1. Introduction Nomura, Sakai, Ishi, & Shirai, 1996). Nineteen variants

of collagen have been reported, named type I–XIX (Bai-
Collagen is an abundant protein in vertebrates and ley, Paul, & Knott, 1998). So far, the main sources of
constitutes 30% of total animal protein (Muyonga, collagen are limited to those of land-based animals, such
Cole, & Duodu, 2004). Tendon, skin, bone, vascular sys- as bovine or porcine skin and bone. However, the out-
tem and connective tissue sheaths surrounding muscle breaks of bovine spongiform encephalopathy (BSE)
are the organs of animals that are mainly structured and the foot-and-mouth disease (FMD) crisis have re-
by collagen (Foegeding, Lanier, & Hultin, 1996). Fur- sulted in anxiety among users of collagen and colla-
thermore, collagen has been found in fish skin, bone gen-derived products of land animal origin (Helcke,
and scale (Ikoma, Kobayashi, Tanaka, Walash, & 2000). Additionally, collagen obtained from porcine
Mann, 2003; Kimura, 1992; Nagai & Suzuki, 2000; skin or bone cannot be used as a component of some
foods due to aesthetic and religious objections
*
Corresponding author. Tel.: +66 74 286334; fax: +66 74 212889. (Sadowska, Kolodziejska, & Niecikowska, 2003). There-
E-mail address: [email protected] (S. Benjakul). fore, alternative sources, such as fish processing waste,

0308-8146/$ - see front matter
2004 Published by Elsevier Ltd.

doi:10.1016/j.foodchem.2004.10.026
476 A. Jongjareonrak et al. / Food Chemistry 93 (2005) 475–484

including skin, bone or scale, have received increasing 2.2. Fish skin preparation
attention for collagen extraction.
The fish processing industry is an important income Brownstripe red snapper (Lutjanus vitta), with an
generator for Thailand. During processing, a large average length of 22–25 cm, were caught from the Song-
amount of wastes is generated. Fish solid wastes consti- khla coast along the Gulf of Thailand, stored in ice and
tute 50–70% of the original raw material, depending on off-loaded 24–36 h after capture. Upon arrival at the
the processes used and types of products. Those wastes dock in Songkhla, fish were stored in ice with a fish/ice
have been of interest as high-protein human foods, in- ratio of 1:2 (w/w) and transported to the Department
stead of employing them as pet foods (Montero, Jimen- of Food Technology, Prince of Songkla University,
nez-Colmenero, & Borderias, 1991; Shahidi, 1994). Hat Yai. Fish were washed using tap water. Skins were
Among the value-added products derived from those then removed, descaled, and cut into small pieces
wastes, collagen from skin, scale and bone has increas- (0.5 · 0.5 cm). Skins were kept on ice prior to collagen
ingly been of interest owing to its abundance. Collagen extraction.
contents (acid-solubilised collagen; ASC) in fish skins
of young and adult Nile perch were 63.1% and 58.7%, 2.3. Skin collagen preparation
respectively (Muyonga et al., 2004), whereas those of
Japanese sea-bass, chub mackerel and bullhead shark The collagen was extracted according to the method of
were 51.4%, 49.8% and 50.1%, respectively (Nagai & Su- Nagai & Suzuki (2000) with a slight modification. All
zuki, 2000). Collagen contents generally vary with fish processes were carried out at 4 C. Skin was soaked in
species, age and season (Ciarlo, Paredi, & Fraga, 1997; 0.1 M NaOH with a sample/solution ratio of 1:30
Foegeding et al., 1996; Montero et al., 1991; Nagai, (w/v) for 24 h with gentle stirring. The solution was chan-
Araki, & Suzuki, 2002). Type I collagen is a fibrous col- ged every 8 h to remove noncollagenous proteins and pig-
lagen and is the major type in fish waste materials, ments. Alkali-treated skins were then washed with
including skin, bone, scale and fins of various fish spe- distilled water until neutral or faintly basic pHs of wash
cies (Ikoma et al., 2003; Kimura, 1983; Kimura, 1992; water were obtained. Fat was removed in 10% (v/v) butyl
Nagai & Suzuki, 2000; Nagai et al., 2002; Sato, Yoshi- alcohol with a sample/solution ratio of 1:30 (w/v) for 24 h
naka, Itoh, & Sato, 1989). Recently, biochemical prop- with gentle stirring and a change of solution every 8 h.
erties of black drum and sheapshead seabream skin Defatted skins were thoroughly washed with distilled
collagen, (subtropical fish) have been characterised water. The matter was soaked in 0.5 M acetic acid with
(Ogawa et al., 2003). Nevertheless, collagen from differ- a sample/solution ratio of 1:30 (w/v) for 24 h with gentle
ent species and habitats might be different in terms of stirring. The mixture was then centrifuged at 20,000g for
molecular compositions and properties (Foegeding 1 h at 4 C. The supernatants were collected and kept at 4
et al., 1996). So far, little information regarding the C. The precipitate was re-extracted in 0.5 M acetic acid
characteristics of marine fish skin collagen, especially with a sample/solution ratio of 1:30 (w/v) for 16 h with
from commercially important species involving those gentle stirring, followed by centrifugation at 20,000g
used for surimi production, has been reported. There- for 1 h at 4 C. The supernatants obtained were combined
fore, this investigation aimed to isolate and characterise with the first extract. The combined extracts were precip-
acid- and pepsin-solubilised collagens (PSC) from the itated by the addition of NaCl to a final concentration of
skin of Brownstripe red snapper (Lutjanus vitta) which 2.6 M in 0.05 M Tris–HCl (pH 7.5). The resultant precip-
is one of the main fish species used for surimi production itate was collected by centrifugation at 20,000g for 1 h at
in Thailand. 4 C and then dissolved in 10 volumes of 0.5 M acetic
acid. The solution obtained was dialysed with 10 volumes
of 0.1 M acetic acid in a dialysis membrane with molecu-
2. Materials and methods lar weight cut-off of 30 kDa for 12 h at 4 C with change
of solution every 4 h. Subsequently, the solution was dia-
2.1. Chemicals lysed with 10 volumes of distilled water with changes of
water until neutral pH was obtained. The dialysate was
b-Mercaptoethanol (bME), pepsin (EC 3.4.23.1) freeze-dried and referred to as ASC. Undissolved residue,
powdered; 750 U/mg dry matter, Staphylococcus aureus obtained after acid extraction, was thoroughly rinsed
V8 protease (EC 3.4.21.19) and protein markers were with distilled water, suspended in 2 volumes of 0.5 M ace-
purchased from Sigma Chemical Co. (St. Louis, Mo, tic acid and subjected to limit hydrolysis with 10% (w/v)
USA). Sodium dodecyl sulfate (SDS), acetic acid, and pepsin (EC 3.4.23.1; powderised; 750 U/mg dry matter,
Tris(hydroxymethyl)aminomethane were obtained from Sigma Chemical Co. (St. Louis, Mo, USA)) for 48 h at
Merck (Darmstadt, Germany). Achromobacter lyticus 4 C with gentle stirring. The viscous solution was centri-
Lysyl endopeptidase (EC 3.4.21.50) was from Wako fuged at 20,000g for 1 h at 4 C. To terminate the pepsin
Pure Chemical Industries, Ltd. (Tokyo, Japan). reaction, the supernatant obtained was dialysed against
 A. Jongjareonrak et al. / Food Chemistry 93 (2005) 475–484 477

10 volumes of 0.02 M sodium phosphate buffer (pH 7.2) 2.6. Peptide mapping of collagen
in a dialysis membrane with molecular weight cut off of
30 kDa for 24 h at 4 C with change of solution every 4 Peptide mapping of collagen samples was performed
h. The dialysate obtained was centrifuged at 20,000g according to the method of Saito, Kunisaki, Urano, &
for 1 h. The pellet obtained was dissolved in 10 volumes Kimura (2002) with a slight modification. The freeze-
of 0.5 M acetic acid. The solution was further precipi- dried samples (0.2 mg) were dissolved in 0.1 ml of 0.1
tated by the addition of NaCl to a final concentration M sodium phosphate, pH 7.2 containing 0.5% (w/v)
of 2.6 M in 0.05 M Tris–HCl (pH 7.5). The resultant pre- SDS. After the addition of 10 ll of the same buffer con-
cipitate was collected by centrifugation at 20,000g for 1 h taining 5 lg of S. aureus V8 protease (EC 3.4.21.19, 800
at 4 C and re-dissolved in 10 volumes of 0.5 M acetic unit/mg solid Sigma Chemical Co., St. Louis, Mo.,
acid. The solution was dialysed with distilled water and U.S.A.) or 0.05 lg of lysyl endopeptidase from A. lyticus
freeze-dried in the same manner as for ASC preparation. (EC 3.4.21.50; 4.5 AU/mg protein; Wako Pure Chemical
Dry matter was referred to as PSC. Industries, Ltd., Tokyo, Japan) to collagen solutions,
the reaction mixture was incubated at 37 C for 25
min or 5 min for V8 protease and lysyl endopeptidase,
2.4. Electrophoretic analysis
respectively. The reaction was terminated by subjecting
the reaction mixture to boiling water for 3 min. Peptides
Protein patterns of collagen samples were analysed
generated by the protease digestion were separated by
using sodium dodecyl sulfate–polyacrylamide gel elec-
SDS–PAGE using 7.5% gel. Peptide mapping of calf
trophoresis (SDS–PAGE) according to the method of
skin collagen acid-soluble type I was conducted in the
Laemmli (1970). Collagen samples were dissolved in
same manner and the peptide maps were compared.
0.02 M sodium phosphate buffer (pH 7.2) containing
1% (w/v) SDS and 3.5 M urea. The sample mixtures
2.7. Collagen solubility test
were gently stirred at 4 C for 12 h to dissolve total
proteins. Supernatants were collected after centrifuging
2.7.1. Solubility determination
at 3000g for 3 min at 4 C. Solubilised collagen
The solubility of collagens was determined in 0.5 M
samples were mixed with the sample buffer (0.5 M
acetic acid at various pH levels and NaCl concentrations
Tris–HCl, pH 6.8 containing 4% (w/v) SDS, 20%
according to the method of Montero et al. (1991) with a
(v/v) glycerol) with and without 10% (v/v) bME, using
slight modification. Collagen samples were dissolved in
the sample/sample buffer ratio of 1:1 (v/v). Samples
0.5 M acetic acid with gentle stirring at 4 C for 12 h
were loaded on to the PAGEL-Compact precast gel
to obtain the final concentrations of 3 and 6 mg/ml.
(5% gel) and subjected to electrophoresis at a constant
current of 20 mA/gel using a Compact-PAGE appara-
2.7.2. Effect of pHs on collagen solubility
tus (Atto Co., Tokyo, Japan). After electrophoresis, gel
Eight ml of collagen solutions (3 mg/ml) were trans-
was stained with 0.05% (w/v) Coomassie blue R-250 in
ferred to a centrifuge tube and the pH was adjusted with
15% (v/v) methanol and 5% (v/v) acetic acid and de-
either 6 M NaOH or 6 M HCl to obtain a final pH rang-
stained with 30% (v/v) methanol and 10% (v/v) acetic
ing from 1 to 10. The volume of sample solutions was
acid. High-molecular-weight markers (Sigma Chemical
made up to 10 ml with distilled water, previously ad-
Co., St. Louis, Mo, USA) were used to estimate the
justed to the same pH as the collagen sample solutions
molecular weights of proteins. Calf skin acid-soluble
tested. The solutions were stirred gently for 30 min at
type I collagen (Sigma Chemical Co., St. Louis, Mo,
4 C and centrifuged at 10,000g for 30 min at 4 C. Pro-
USA), porcine cartilage acid-soluble type II collagen,
tein content in the supernatants was determined by the
porcine skin acid-soluble type III collagen, and porcine
method of Lowry, Rosebrough, Farr, & Randall
placenta acid-soluble type V collagen (Wako Pure
(1951) using bovine serum albumin as a protein stan-
Chemical Industries, Ltd., Tokyo, Japan) were used
dard. Relative solubility of collagen samples was calcu-
as standard collagens.
lated in comparison with that obtained at the pH
rendering the highest solubility.
2.5. Amino acid composition
2.7.3. Effect of salt concentration on collagen solubility
Collagen samples were hydrolysed under reduced Five ml of collagen solutions (6 mg/ml) in 0.5 M ace-
pressure in 4.0 M methanesulfonic acid containing tic acid were mixed with 5 ml of cold NaCl in acetic acid
0.2% (v/v) 3-2(2-aminoethyl)indole at 115 C for 24 h, of various concentrations (0%, 2%, 4%, 6%, 8%, 10%
and the hydrolysates were neutralised with 3.5 M NaOH and 12% (w/v)), to obtain the final NaCl concentrations
and diluted with 0.2 M citrate buffer (pH 2.2). An ali- of 1%, 2%, 3%, 4%, 5% and 6% (w/v). The mixtures were
quot of 0.4 ml was applied to an amino acid analyser stirred gently at 4 C for 30 min and centrifuged at
(MLC-703; Atto Co., Tokyo, Japan). 10,000g for 30 min at 4 C. Protein content in the
478 A. Jongjareonrak et al. / Food Chemistry 93 (2005) 475–484

supernatants was determined by the method of Lowry at the telopeptide region were cleaved without damaging
et al. (1951) and relative solubility was calculated in the integrity of the triple helix. Therefore, the collagen
comparison with that found at the salt concentration with the predominant monomeric molecules could be
exhibiting the highest solubility. solubilised in the presence of acid (Hickman et al.,
2000). From the result, the major fraction of collagen
2.8. Thermal transition measurement from Brownstripe red snapper skin was ASC (66% based
on extractable collagen weight) and a lower content of
Collagen samples were prepared by the method de- PSC was found (34%). The greater content of ASC frac-
scribed by Rochdi, Foucat, & Renou (2000) with a slight tion in Brownstripe red snapper skin was in accordance
modification. The freeze-dried collagen samples were with those reported in bigeye snapper (Priacanthus
rehydrated in deionised water or 0.05 M acetic acid solu- marcracanthus) skin (85%) (Jongjareonrak et al., ac-
tion with a sample/solution ratio of 1:40 (w/v). The mix- cepted) and hake (Merluccius hubbsi) skin (85%) (Ciarlo
tures were allowed to stand for 2 days at 4 C. et al., 1997). However, the PSC fraction in Brownstripe
The thermal transition of collagens was measured red snapper skin was 2-fold higher than those obtained
using Perkin–Elmer Differential Scanning Calorimetry from those two species. It is suggested that collagen with
(DSC) (Model DSC-7, Norwalk, CT, USA). Tempera- more inter-molecular cross-links is present to a greater
ture calibration was performed using the Indium ther- extent in the skin of Brownstripe red snapper, than in
mogram. The rehydrated samples (5–10 mg) were bigeye snapper and hake skin.
accurately weighed into aluminium pans, sealed, and
scanned over the range of 20–50 C with a heating rate 3.2. Sodium dodecyl sulfate–polyacrylamide gel
of 1C/min. Ice water was used as a cooling medium and electrophoresis
the system was equilibrated at 20 C for 5 min prior to
the scan. The empty aluminium pan was used as the ref- The electrophoretic patterns of ASC and PSC were
erence. The maximum transition temperature (Tmax) was analysed in the presence and absence of bME (Fig. 1).
estimated from the maximum peak of DSC transition Generally, no differences in the electrophoretic patterns
curve. of ASC or PSC, with or without bME, were observed,
indicating that both collagen fractions contained no
2.9. Statistical analysis disulfide bonds. Montero, Borderias, Turnay, & Ley-
zarbe (1990) reported that similar electrophoretic dia-
Data were subjected to analysis of variance (ANO- grams of collagen from the skin of both hake and
VA). Comparison of means was carried out by DuncanÕs trout were observed in the presence or absence of
multiple-range test (Steel & Torrie, 1980). Analysis was bME. From this result, a1- and a2-chains were found
performed using a SPSS package (SPSS 8.0 for win- as the major constituents for both ASC and PSC.
dows, SPSS Inc, Chicago, IL). High-molecular-weight components (High MW compo-
nents), including b- and c-components, as well as their
cross-linked molecules, were also observed in both
3. Results and discussion fractions.
High MW cross-linked molecules in collagen increase
3.1. Isolation of ASC and PSC from Brownstripe red with animal age (Foegeding et al., 1996) and starving
snapper skin fish has more cross-linked collagen than those that are
well fed (Love, Yamaguchi, Creach, & Lavety, 1976;
Acid-solubilised collagen and pepsin-solubilised col- Sikorski, Kolakowska, & Pan, 1990). However, the
lagen were isolated from Brownstripe red snapper skin cross-linking rate of collagen in fish skins is extremely
with yields of 9.0% and 4.7% (wet weight basis), respec- slow and the highly cross-linked molecule is rarely found
tively. The skin was not completely solubilised by 0.5 M (Cohen-Solal, Louis, Allian, & Meunier, 1981). High
acetic acid extraction. This result was in agreement with MW cross-linked molecules in ASC were possibly di-
Jongjareonrak, Benjakul, Visessanguan, & Tanaka (ac- gested, as evidenced by the lower density of b- and c-
cepted) who reported the incomplete solubilisation of component bands with a concomitant increase in band
bigeye snapper skin in 0.5 M acetic acid. The result sug- intensity of a-chains in the PSC fraction. Pepsin cleaves
gested that the collagen molecules in Brownstripe red the cross-link containing telopeptide, and b-chain is con-
snapper skin were most likely cross-linked by covalent comitantly converted to two a-chains (Sato et al., 2000).
bonds through the condensation of aldehyde groups at The band intensity of the a1-chain was 2-fold higher
the telopeptide region as well as the inter-molecular than that of the a2-chain for both ASC and PSC. The
cross-linking, leading to a decrease in solubility of colla- a1- and a2-chain patterns were similar to that of stan-
gen (Burghagen, 1999; Foegeding et al., 1996). With fur- dard collagen type I from calf skin (Lane 2). It is sug-
ther limited pepsin digestion, the cross-linked molecules gested that type I collagen is the major collagen in
 A. Jongjareonrak et al. / Food Chemistry 93 (2005) 475–484 479

Fig. 1. Protein pattern of ASC and PSC from Brownstripe red snapper skin under reducing and non-reducing conditions. Lane 1: high MW protein
markers; lanes 2, 3, 8, and 9: collagen types I, II, III, and V, respectively; lanes 4 and 5: ASC and PSC under non-reducing conditions; lanes 6 and 7,
ASC and PSC under reducing conditions.

both ASC and PSC from Brownstripe red snapper skin. Table 1
This observation is in agreement with the findings re- Amino acid composition of ASC and PSC from Brownstripe red
snapper skin (residues per 1000 total amino acid residues)
ported for skin collagens from hake (Ciarlo et al.,
1997; Montero et al., 1990), trout (Montero et al., Amino acids ASC PSC
1990), Nile perch (Muyonga et al., 2004), blackdrum Hydroxyproline 81 86
and sheephead seabream (Ogawa et al., 2003) and bigeye Aspartic acid 50 49
Threonine 29 30
snapper (Kittiphattanabawon, Benjakul, Visessanguan, Serine 37 39
Nagai, & Tanaka, 2005). Type I collagen consists of Glutamic acid 81 79
two a1- and one a2-chain as the major component Proline 131 135
([a1]2a2). Since the a3-chain has a molecular mass indis- Glycine 252 235
tinguishable from a1 and as it cannot be separated from Alanine 143 142
Valine 18 17
a1 under the electrophoretic conditions employed, the Methionine 15 14
co-presence of a3 with a1 might be possible. Isoleucine 7 8
Leucine 24 24
3.3. Amino acid composition Tyrosine 4 2
Phenylalanine 15 16
Hydroxylysine 9 15
The amino acid composition of ASC and PSC is ex- Lysine 33 34
pressed as residues per 1000 total amino acid residues Histidine 7 6
and is shown in Table 1. Generally, ASC and PSC ex- Arginine 65 68
tracted from Brownstripe red snapper skin had similar Total 1000 1000
amino acid profiles. From the results, ASC and PSC
were rich in glycine (25.2% and 23.5%), alanine
(14.3% and 14.2%), and proline (13.1% and 13.5%). in collagen. The amounts of imino acids, proline and
In general, glycine occurs uniformly, at every third res- hydroxyproline, in ASC and PSC were 212 and 221
idue throughout most of collagen molecules, except for residues per 1000 residues, respectively. The contents
the first 14 amino acids from the N-terminus and the of imino acids in ASC and PSC from Brownstripe
first 10 from the C-terminus (Burghagen, 1999; Foege- red snapper skin were relatively high and were similar
ding et al., 1996; Wong, 1989). Both the ASC and the to that of ASC from calf skin (215 residues per 1000
PSC fraction consisted of proline, hydroxyproline and residues) (Herbage, Bouillet, & Bernengo, 1977). Imino
hydroxylysine, which are unique amino acids found acids contribute to the denaturation temperature and
480 A. Jongjareonrak et al. / Food Chemistry 93 (2005) 475–484

the stability of the helix structure of collagen (Ikoma 3.4. Peptide mapping of collagen
et al., 2003). ASC and PSC had high levels of hydroxy-
proline and hydroxylysine and were similar to ASC Peptide maps of collagens (ASC and PSC) digested
from calf skin reported by Herbage et al. (1977). This by V8 protease and lysyl endopeptidase, in comparison
result suggested a high degree of oxidation of hydrox- with calf skin collagen type I, were revealed by SDS–
ylated residues of proline and lysine in collagens from PAGE (7.5% gel) as shown in Fig. 2. Generally, band
Brownstripe red snapper skin. The formations of intensity of a1- and a2-chains, as well as high MW
hydroxyproline and hydroxylysine were catalysed by cross-link, c- and b-components of ASC, PSC and calf
proline hydroxylase and lysine hydroxylase, respec- skin collagen type I, decreased after limited digestion
tively (Foegeding et al., 1996; Wong, 1989). Imino acid by V8 protease (Lanes 5–7) and lysyl endopeptidase
contents of collagen from Brownstripe red snapper skin (Lanes 8–10). A concomitant increase in small peptides
were relatively high, compared with those reported for was also observed. Calf skin collagen type I digested by
collagen from skins of carp, hake, trout, ocellate puffer, V8 protease (Lane 5) underwent a slight decrease in
black drum, sheephead and bigeye snapper, which con- band intensity of the a-, b-components, and high
tained imino acids ranging from 158 to 211 residues MW cross-linked molecules and there was an appear-
per 1000 residues (Jongjareonrak et al., accepted; Kim- ance of peptide fragments with MW of 154.7, 95.0,
ura, 1992; Kittiphattanabawon et al., 2005; Montero 83.6 and 38.6 kDa. For ASC and PSC, the a-
et al., 1990; Nagai et al., 2002; Ogawa et al., 2003). component and high MW cross-linked molecules were
Proline and hydroxyproline contents vary with species more hydrolysed after digestion with V8 protease.
and their living habitat (Foegeding et al., 1996; Love The results suggest that the a-component and high
et al., 1976). The pyrolidine rings of proline and MW cross-linked molecules from calf skin collagen
hydroxyproline impose restrictions on the conforma- type I are more tolerant to hydrolysis by V8 protease
tion of the polypeptide chain and help to strengthen than are ASC and PSC from Brownstripe red snapper
the triple helix (Wong, 1989). Thus, collagen from skin. V8 protease shows a high specific preference for
Brownstripe red snapper might have a different molec- glutamic acid and aspartic acid residues of proteins
ular conformation from that of other species owing to (Vercaigne-Marko, Kosciarz, Nedjar-Arroume, & Guil-
the high content of imino acids. lochon, 2000). Due to the lower contents of glutamic

Fig. 2. Peptide maps of ASC and PSC from Brownstripe red snapper skin digested by V8 protease and lysyl endopeptidase. Lanes 1 and 11: high and
low MW protein markers, respectively; lanes 2, 3, and 4: collagen type I, ASC, and PSC; lanes 5, 6, and 7: peptide fragments of collagen type I, ASC,
and PSC with V8 protease digestion, respectively; lanes 8, 9, and 10: peptide fragments of collagen type I, ASC, and PSC with lysyl endopeptidase
digestion, respectively.
 A. Jongjareonrak et al. / Food Chemistry 93 (2005) 475–484 481

acid and aspartic acid residues (75 and 45 residues per 110
1000 residues) in calf skin collagen (Herbage et al., 100 ASC

Relative collagen solubility (%)

1977), ASC (81 and 50 residues per 1000 residues) 90 PSC
and PSC (79 and 49 residues per 1000 residues), which
80
had the greater glutamic acid and aspartic acid con-
tents, might be more susceptible to hydrolysis by V8 70
protease. From this result, ASC was more prone to 60
hydrolysis than PSC. However, similar patterns of pep-
50
tide fragments were observed. After hydrolysis, the a-
40
component and high MW cross-linked molecules of
ASC from the skin of Brownstripe red snapper were 30
degraded into small MW peptides, ranging from 20
103.6 to 34.0 kDa. In addition, peptide fragments of
10
high MW, ranging from 205 to 116 kDa, were also ob-
served. For PSC, the a-component was hydrolysed to 0
some extent, while high MW cross-linked molecules 0 1 2 3 4 5 6 7 8 9 10
were totally digested and small MW peptides were also pH

observed with molecular weights ranging from 103.6 to Fig. 3. Solubility of ASC and PSC from Brownstripe red snapper skin
32.3 kDa. in 0.5 M acetic acid at different pHs.
For the peptide map of collagens digested by lysyl
endopeptidase (Lanes 8–10), all collagens were more
susceptible to hydrolysis by lysyl endopeptidase than The solubilities of ASC and PSC reached maxima at
by V8 protease, as evidenced by the disappearance of pH 3 and 4, respectively (P < 0.05). In general, both col-
the b-component (205 kDa) and the high MW cross- lagens were solubilised to a greater extent in acidic pH
linked molecules, with a concomitant increase in small ranging from 1 to 4. Marked decreases in solubility were
MW fragments. This result was in accordance with the observed and reached the minimum when the pH was
peptide mapping of collagens from skin and bone of big- increased to 7 (P < 0.05). The further increases in pH
eye snapper, as reported by Kittiphattanabawon et al. up to 10 caused a slight increase in solubility. As the
(2005). The a-component and the high MW cross-linked pH is lower or higher than pI, the net charge residues
molecules of calf skin collagen type I and ASC from of protein molecules are greater and the solubility is in-
Brownstripe red snapper skin mostly disappeared after creased by the repulsion forces between chains (Vojdani,
hydrolysis, while the a-component of PSC still remained 1996). In contrast, total net charges of protein molecules
to some extent. The generated peptides from ASC and are zero and hydrophobic–hydrophobic interaction in-
PSC, after digestion, had MWs ranging from 205 to creases, thereby leading to the precipitation and aggre-
30 kDa, while those of calf skin collagen were lesser in gation at pI. It has been reported that collagen has
number of bands. The differences in peptide map be- isoelectric points at pH 6–9 (Foegeding et al., 1996).
tween the different collagens generated by lysyl endopep- Thus the lowest solubility of ASC and PSC was ob-
tidase and V8 protease digestion suggest that there tained at pH around 7. This result was in accordance
might be some differences in their primary structure, with the solubility of collagen from trout muscle and
especially the a-helix strand (Nagai et al., 2002; Omura, skin, which was lowest at pH 7 (Montero et al., 1991).
Urano, & Kimura, 1996; Yoshinaka, Mizuta, Suzuki, & From the result, ASC was solubilised more than PSC
Sato, 1991). Accessibility of susceptible bonds in colla- at all pHs tested except at pH 2 and 3, suggesting a
gen structure to proteinase might be different, causing higher degree of molecular crosslinking of the ASC frac-
varying degrees of hydrolysis between ASC and PSC. tion, or the predominance of stronger bonds than PSC
Peptide maps of collagens were reported to differ among (Montero, Gomez-Guillen, & Borderias, 1999). This
sources and species (Mizuta, Yamasa, Miyagi, & Yoshi- was evidenced by the higher contents of high MW
naka, 1999). Thus, ASC and PSC from Brownstripe red cross-link of ASC than of PSC (Fig. 1). The variation
snapper skin might be different in terms of domain or in solubility of collagens with pH has been reported
cross-links and totally different from calf skin collagen for bigeye snapper skin and bone over the pH ranges
type I in term of sequence and composition of amino of 1–10 (Kittiphattanabawon et al., 2005).
acids.
3.6. Effect of salt concentration on collagen solubility
3.5. Effect of pHs on collagen solubility
The effect of NaCl on the solubility of ASC and PSC
The effect of pH on the solubility of ASC and PSC extracted from Brownstripe red snapper skin is depicted
from Brownstripe red snapper skin is shown in Fig. 3. in Fig. 4. Solubility of ASC and PSC in 0.5 M acetic acid
482 A. Jongjareonrak et al. / Food Chemistry 93 (2005) 475–484

110 remained constant at NaCl concentrations up to 2%

ASC (P > 0.05). A drastic decrease in the solubility of ASC
Relative collagen solubility (%)

100
PSC was observed at 3% NaCl or above (P < 0.05). For
90
80 PSC, a slight decrease in solubility was obtained in the
presence of 3% NaCl. A sharp decrease in solubility
70
was observed with 4% NaCl or above. The solubility
60
of collagens from the skin of trout, hake, bigeye snapper
50
(Priacanthus tayenus), and bigeye snapper (P. marcr-
40 acanthus) in acetic acid solution generally decreased with
30 increasing NaCl concentration (Kittiphattanabawon
20 et al., 2005; Jongjareonrak et al., accepted; Montero
10 et al., 1999, 1991). The decrease in solubility of collagens
0 could be described by the salting out phenomenon which
0 1 2 3 4 5 6 occurred at relatively low NaCl concentrations (Asghar
Concentration of NaCl (%(w/v)) & Henrickson, 1982). An increase in ionic strength
causes a reduction in protein solubility by an enhanced
Fig. 4. Solubility of ASC and PSC from Brownstripe red snapper skin
hydrophobic–hydrophobic interaction between protein
in 0.5 M acetic acid with different NaCl concentrations.

Tm= 30.52 o C
0.05 M acetic acid

Tm= 31.52 o C
Heat Flow

deionised water

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
(a) Temperature (ºC)

Tm= 30.46 o C
Heat Flow

0.05 M acetic acid

Tm= 31.02 o C
deionised water

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
(b) Temperature (ºC)
Fig. 5. Thermograms of ASC (a) and PSC (b) from Brownstripe red snapper skin rehydrated in 0.05 M acetic acid and deionised water.
 A. Jongjareonrak et al. / Food Chemistry 93 (2005) 475–484 483

chains, and the competing for water of ionic salts, lead- crease in the transition temperature of collagens rehy-
ing to the induced protein precipitation (Damodaran, drated in 0.05 M acetic acid, as observed by the lower
1996; Vojdani, 1996). From the result, similar behaviour Tmax of both ASC and PSC.
was found for both collagen fractions. However, PSC
exhibited a greater solubility than ASC at NaCl concen-
trations greater than 2%. A greater solubility of PSC 4. Conclusion
could be due to the partial hydrolysis of high MW
cross-linked molecules by pepsin. In addition, the differ- ASC and PSC from the skin of Brownstripe red snap-
ences in compositions and molecular species between per were isolated with yields of 9% and 4.7%, respec-
ASC and PSC fractions might result in such different tively. The collagens were characterised as type I
characteristics. without disulfide bond. ASC exhibited some differences
in amino acid profiles, protein patterns, and sequence of
3.7. Thermal stability of collagen primary structure, compared to PSC and was totally dif-
ferent from calf skin collagen. Denaturation tempera-
Thermal transitions of ASC and PSC from Brown- tures of collagens were similar between ASC and PSC
stripe red snapper skin rehydrated in deionised water and relatively higher than that of temperate fish. ASC
and 0.05 M acetic acid are depicted in Fig. 5. ASC and PSC were soluble at acidic pH and lost the solubility
and PSC showed transition curves with maximum tem- with increasing salt concentrations.
peratures (Tmax) of 31.52 and 31.02 C, respectively, in
deionised water and at 30.52 and 30.46 C, respectively,
in 0.05 M acetic acid. Tmax of ASC was similar to that of Acknowledgements
PSC in both media, suggesting no differences in the
denaturation temperature between the two fractions. This research was supported by the Thailand Re-
The triple helix structure was still predominant in the search Fund under The Royal Golden Jubilee PhD pro-
PSC fraction when the skin material was limited to gramme to Akkasit Jongjareonrak (PHD/0060/2544).
digestion by pepsin (Hickman et al., 2000). Tmax of Authors thank Professor Akira Shinagawa of Environ-
ASC and PSC from Brownstripe red snapper skin was ment Education Center, Gakushuin WomenÕs College
much lower than that of mammalian collagen, such as (Tokyo, Japan) for amino acid analysis.
calf skin collagen (37 C) (Ogawa et al., 2003). Imino
acid contents (proline and hydroxyproline) show a di-
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