Food Bioscience 9 (2015) 68 –74

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/fbio

Extraction and characterization of acid- and

pepsin-soluble collagens from the scales, skins and
swim-bladders of grass carp (Ctenopharyngodon idella)

Dasong Liua,b, Xuan Zhanga, Tiancheng Lia, Hongxu Yanga,

Hongchao Zhanga, Joe M. Regensteinc, Peng Zhoua,b,n
a
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University,
Wuxi, Jiangsu Province 214122, China
b
Synergetic Innovation Center of Food Safety and Nutrition, Wuxi, Jiangsu Province 214122, China
c
Department of Food Science, Cornell University, Ithaca, NY 14853-7201, USA

ar t ic l e in f o abs tra ct

Article history: The objective of this study was to characterize and compare the acid-soluble collagen (ASC)
Received 4 September 2014 and pepsin-soluble collagen (PSC) from the scales, skins and swim-bladders of grass carp.
Accepted 12 December 2014 Both ASC and PSC from the three tissues were mainly characterized as type I collagen, with
Available online 20 December 2014 similar electrophoretic patterns and amino acid profiles. PSC from the three tissues was

Keywords: more susceptible to hydrolysis by V8 protease than ASC. Both ASC and PSC from the three

Acid-soluble collagen tissues showed similar peptide hydrolysis patterns. The maximum transition tempera-

Pepsin-soluble collagen tures of both ASC and PSC from the internal tissue (swim-bladders) were only slightly

Grass carp higher than those of ASC and PSC from the external tissues (scales and skins). These

Scale results suggest that collagens from the three tissues of grass carp had almost similar

Skin biochemical properties, and could be used as potential substitutes for mammalian

Swim-bladder collagens due to their comparatively high thermostability.

& 2014 Elsevier Ltd. All rights reserved.

1. Introduction connective tissues where they play a role in maintaining

Collagen, the major structural protein in both vertebrates and
invertebrates, constitutes about 30% of the total animal's
proteins (Muyonga, Cole, & Duodu, 2004). Currently, at least
29 distinct types of collagens, which are produced by more
than 30 genes, have been identified (McCormick, 2009). Each
type of collagen differs considerably in its sequence, structure
and function, and thus they are specifically distributed in the
skins, bones, tendons, vascular systems or intramuscular
the stability and structural integrity of the corresponding
tissues and organs.
Type I collagen is identified as the major fibrillar collagen
in almost all vertebrae connective tissues. It is unique in
forming a right-handed triple superhelical structure consist-
ing of three similarly sized left-handed helical polypeptide
chains. The collagen is also characterized by a Gly-X–Y
repeating motif with the X and Y positions being mostly
occupied by Pro and Hyp, respectively (Gelse, Pöschl, &

n
Corresponding author at: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan
University, 1800 Lihu Avenue, Wuxi, Jiangsu Province 214122, China. Tel./fax: þ86 510 85912123.
E-mail address: zhoupeng@jiangnan.edu.cn (P. Zhou).

http://dx.doi.org/10.1016/j.fbio.2014.12.004
2212-4292/& 2014 Elsevier Ltd. All rights reserved.
 Food Bioscience 9 (2015) 68 –74
Aigner, 2003; Liu, Regenstein, Mulvaney, Boran, & Zhou, 2013).
Purified type I collagen can self-assemble into native-like
collagen fibril in vitro with high tensile strength and stability,
suggesting its potential application in the development of
novel biomimetic material used for treating burns and
wounds. In addition, some food-derived collagen peptides,
such as Pro-Hyp, have been demonstrated to promote the
proliferation of fibroblasts and the biosynthesis of collagen in
the skin in a collagen-specific manner, by which collagen
exerts its beneficial biological effects on skin conditions (e.g.,
cutaneous anti-aging) after oral ingestion (Zague, 2008).
Therefore, collagen and collagen-derived products have been
widely used in the food, pharmaceutical and cosmetic indus-
tries due to their excellent biocompatibility, easy biodegrad-
ability and weak antigenicity (Liu, Li, Miao, & Wu, 2009).
Traditionally, collagen has been produced from the skins
and bones of land-based animals, such as cow, pig and
poultry. In recent years, the outbreaks of bovine sponge
encephalopathy, foot-and-mouth disease and avian influenza
have led to a negative attitude among some health-conscious
consumers toward these traditional collagen and collagen-
derived products. Additionally, collagens obtained from pig
and other animals that are not religiously slaughtered are not
acceptable for use in kosher and halal food products
(Regenstein, Chaudry, & Regenstein, 2003). Among the alter-
native sources of collagen, fish provides the desired raw
materials such as scales, skins and swim-bladders, owning
to their high abundance of collagens (Niu et al., 2013;
Regenstein & Zhou, 2007; Yang et al., 2007). In the fish
processing industry, a large amount of these by-products
are produced, as they are a major component of the total
by-products that account for 50–70% of the catch weight
(Kittiphattanabawon, Benjakul, Visessanguan, Nagai, & Tanaka,
2005). Most of these by-products are currently used as relatively
low-value feedstuffs or fertilizers (Li et al., 2013). Efficient use
of these by-products can produce value-added products, avoid
environmental problems and even create new job/business
opportunities.
Once secreted into the extracellular matrix, collagen
molecules align head-to-tail in a quarter staggered array that
allows for the occurrence of inter-molecular crosslinking at
the telopeptide, and the extent of crosslinking usually
increases with age (McCormick, 2009). Based on the extrac-
tion methods, collagens can be grouped into salt-, acid- and
pepsin-soluble collagens. Salt-soluble collagen refers to the
newly synthesized and un-crosslinked collagens in tissues,
and the extraction with a cold neutral salt solution usually
leads to a low collagen yield and purity. An acid treatment
can solubilize not only collagen monomers but also collagen
aggregates having some inter-chain cross-linkages such as
the reducible aldimine condensation crosslinks. Limited pro-
teolysis at the telopeptide is used to facilitate solubilization of
collagens that are highly crosslinked by nonreducible triva-
lent bonds at the telopeptide and hard to extract with acid
process alone, and this extraction method will not affect the
structural integrity of collagen super triple-helix (Regenstein
& Zhou, 2007).
Previous research has reported the extraction and character-
ization of collagens from the by-products of many marine fish
species, such as the skins of unicorn leatherjacket (Ahmad &
69
Benjakul, 2010), the skins of Brownstripe red snapper
(Jongjareonrak, Benjakul, Visessanguan, Nagai, & Tanaka,
2005) and the scales of spotted golden goatfish (Matmaroh,
Benjakul, Prodpran, Encarnacion, & Kishimura, 2011). With the
decline in the harvest of sea-water fish species, much attention
has been paid to using the processing by-products from fresh-
water fish species as the potential alternative collagen sources.
In China, grass carp is one of the “four major cultured
fresh-water fish species” along with black carp, bighead carp
and silver carp (Wang, Yang, Wang, & Du, 2008b). In previous
studies, collagens from different tissues of each of these
four carp species have been investigated separately, and
comparison of results from these separate studies showed
great variations in the biochemical and functional properties
of the collagens. For example, the denaturation tempera-
tures of scale collagen (Zhang, Duan, Ye, & Konno, 2010) and
skin collagen (Rodziewicz-Motowidło et al., 2008) from silver
carp were reported to be 29.0 and 34.8 1C, respectively. In
addition, the scale collagen (Li et al., 2008) and skin collagen
(Zhang et al., 2007) from grass carp showed denaturation
temperatures of 32.0 and 24.6 1C, respectively. However, as
the extraction procedures and the analysis methods used in
those independent studies varied, it is uncertain whether
such results truly reflect the in vivo variations, if present, of
collagens from the different tissues of one fish. Therefore,
comparison and interpretation of results from different
research groups should be done with caution. Thus, this study
was conducted to extract and characterize ASC and PSC from
the scales, skins and swim-bladders of grass carp with care-
fully designed extraction procedures and analysis methods to
provide a more robust comparison of collagens from the three
tissues of one fish.
2. Materials and methods
2.1. Chemicals
Pepsin from porcine gastric mucosa (EC 3.4.23.1, 2  crystallized
and lyophilized, 2800 U/mg dry matter based on hemoglobin
hydrolysis) and Staphylococcus aureus V8 protease (EC 3.4.21.19,
lyophilized, 500–1000 U/mg based on the hydrolysis of N-t-Boc-L-
glutamic acid α-phenyl ester) were purchased from Sigma-
Aldrich Co. (St. Louis, MO, USA). The protein standard (GE
Healthcare UK Ltd., Amersham, Buckinghamshire, UK) used for
electrophoretic analysis of ASC and PSC consisted of the follow-
ing proteins: myosin, 220 kDa; α2-macroglobulin, 170 kDa;
β-galactosidase, 116 kDa; transferrin, 76 kDa; glutamic dehydro-
genase, 53 kDa. Precision Plus Protein All Blue Standards (Bio-Rad
Laboratories, Inc., Hercules, CA, USA) used for analysis of the
peptide hydrolysis patterns of ASC and PSC contained a cocktail
of recombinant proteins with the manufacturer's specified
molecular weight of 37, 50, 75, 100, 150 and 250 kDa. All the
other chemicals used were of analytical grade.
2.2. Preparation of fish scales, skins and swim-bladders
Live farmed grass carps, with weights ranging from 3 to 4 kg,
were purchased in the spring from a local market in
Wuxi, Jiangsu Province, China. They were transported to the
70 Food Bioscience 9 (2015) 68 –74
laboratory in water within 30 min. Upon arrival, the fish were
stunned by a sharp blow to the head with a wooden stick in a
walk-in chill room at a temperature around 4 1C. The scales,
skins and swim-bladders were manually removed with a
filleting knife and then washed with cold distilled water.
The cleaned skins and swim-bladders were cut into small
pieces (0.5  0.5 cm2) using a scissor. All the prepared samples
were kept on ice prior to collagen extraction.
2.3. Extraction of ASC and PSC from the scales, skins and
swim-bladders
The extraction of ASC and PSC was done according to the
methods of Liu, Liang, Regenstein, & Zhou (2012) and Heu
et al. (2010) with slight modifications. All procedures were
done in a walk-in chill room at a temperature around 4 1C,
and the solutions were stirred using magnetic stirrers (IKA
Werke GmbH & Co. KG, Staufen, Germany) during pretreat-
ment and extraction.
To remove non-collagenous proteins and pigments, the
scales, skins and swim-bladders were soaked in 20 volumes of
0.1 M NaOH for 36 h, with the alkaline solution being changed
every 12 h. After being thoroughly washed with cold distilled
water, the scale residues were decalcified with 10 volumes of
0.5 M EDTA for 72 h with the solution being changed every
24 h, while the skin and swim-bladder residues were sus-
pended in 20 volumes of 10% (v/v) butyl alcohol for 24 h to
remove fat with a change of solution every 12 h. After being
washed with cold distilled water, the residues were extracted
for 72 h with 40 volumes (skins and swim-bladders) or 10
volumes (scales) of 0.5 M acetic acid to produce ASC or 0.5 M
acetic acid containing 0.1% (w/v) pepsin to produce PSC.
The suspensions were centrifuged at 10,000g for 20 min at
4 1C using an Avanti J-E centrifuge (Beckman Colter, Inc.,
Indianapolis, IN, USA), and the collagens in the supernatants
were salted-out by adding NaCl to a final concentration of
2.0 M. After 12 h, the precipitates were collected by centrifuga-
tion at 10,000g for 20 min and then re-dissolved in 0.5 M acetic
acid. The resulting solutions were dialyzed against cold dis-
tilled water using a dialysis bag with a molecular weight cut-
off of 7 kDa (Shanghai Green Bird Science and Technology
Development Co., Shanghai, China) and then lyophilized using
the Labconco freeze dryer (Labconco Corp., Kansas, MO, USA).
2.4. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE)
SDS-PAGE was done with a Mini-PROTEAN Tetra Cell system
(Bio-Rad Laboratories, Inc.) according to the method of
Laemmli (1970), using a 4% stacking gel and a 5% resolving
gel. ASC or PSC was dissolved in 1% (w/v) SDS to obtain a final
concentration of 2 mg/ml and then mixed with an equal
volume of sample buffer (62.5 mM Tris–HCl, pH 6.8, contain-
ing 2% SDS and 25% (v/v) glycerol). A 10 μl sample was loaded
in each well. After electrophoresis, the gels were stained
with 0.1% (w/v) Coomassie Brilliant Blue R-250 in 50% (v/v)
methanol and 6.8% (v/v) glacial acetic acid for 5 h, and then
destained using 7.5% (v/v) glacial acetic acid and 5%
(v/v) methanol for about 9 h with the solution being changed
every 3 h.
2.5. Amino acid analysis
A 100 mg sample of ASC or PSC was hydrolyzed in 8 ml of 6 M
HCl in an evacuated and sealed tube at 110 1C for 22 h.
An Agilent 1100 series high performance liquid chromatogra-
phy system (Agilent Technologies, Inc., Santa Clara, CA, USA)
equipped with a C18 ODS HYPERSIL column (250 mm  4.6 mm
i.d., 5 mm particle size; Agilent Technologies, Inc.) was used to
analyze the composition of amino acids that were subjected to
pre-column derivatization with o-phthalaldehyde and fluore-
nylmethyl chloroformate. The content of each amino acid was
calculated based on the area of the corresponding peak on the
elution curves of the sample and standard (Sigma-Aldrich Co.),
and the results were expressed as residues per 1000 total
residues recovered by the amino acid analyzer.
2.6. Peptide hydrolysis patterns
Peptide hydrolysis patterns of ASC or PSC were studied
according to the method of Saito, Kunisaki, Urano, &
Kimura (2002) with slight modifications. Samples (0.2 mg)
were dissolved in 0.1 ml of 0.1 M sodium phosphate buffer
(pH 7.2) containing 0.5% (w/v) SDS, and 10 μl of the same
buffer containing 5 μg of S. aureus V8 protease was added to
the protein solution. The mixture was incubated at 37 1C for
25 min and then the reaction was terminated by placing the
test tubes in boiling water for 3 min. The resulting peptides
were separated by SDS-PAGE, using a 4% stacking gel and a
7.5% resolving gel.
2.7. Differential scanning calorimetry (DSC)
DSC studies were done using the Q2000 Series DSC (TA
Instruments, Inc., New Castle, DE, USA) which was calibrated
for temperature and enthalpy using indium as the standard.
ASC or PSC was rehydrated in 40 volumes of 0.05 M acetic
acid for 48 h at 4 1C. The rehydrated samples (10.070.5 mg)
were accurately weighed into aluminum pans (TA Instru-
ments, Inc.), hermetically sealed and scanned from 20 to
50 1C at a rate of 1 1C/min. An empty sealed aluminum pan
was used as the reference. The maximum transition tem-
perature (Tmax) was recorded as the peak temperature of each
endothermic peak.
2.8. Statistical analysis
Statistical analysis was done using SAS of version 8.0 (1999,
SAS Institute, Inc., Cary, NC, USA). Analysis of variance using
the General Linear Model procedure and the difference
between means using the Duncan test were determined at
an α level of 0.05.
3. Results and discussion
3.1. SDS-PAGE patterns
Fig. 1 shows the electrophoretic patterns of ASC and PSC from
the skins, swim-bladders and scales of grass carp. ASC from
these three tissues showed similar electrophoretic patterns,
 Food Bioscience 9 (2015) 68 –74
Fig. 1 – SDS-PAGE patterns of ASC (1, 3 and 5) and PSC (2, 4
and 6) from the skins (1 and 2), swim-bladders (3 and 4) and
scales (5 and 6) of grass carp.
with the major bands being α- and β-chains (dimers of the
α-chains). The band intensity of the α1-chain was approxi-
mately twofold higher than that of the α2-chain. The α3-chain,
if present, might not be separated by the electrophoretic
conditions employed in this study in that it had a similar
molecular weight to that of the α1-chain (Kittiphattanabawon
et al., 2005). In addition, other cross-linked components with
higher molecular weight were also observed in all ASC. For
PSC from the three tissues, similar electrophoretic patterns
were also observed, and the major components were α1- and
α2-chains, with the band intensity ratio also being about 2:1.
The mobility of α-chains of all PSC was slightly higher than
that of α-chains of the corresponding ASC. Moreover, the
band intensity of the β-chains and other highly cross-linked
components from PSC was significantly lower than that of
ASC. Pepsin is able to cleave the cross-linked regions at the
telopeptide without affecting the structural integrity of the
super triple-helix of collagen (Kittiphattanabawon, Benjakul,
Visessanguan, & Shahidi, 2010). Therefore, slightly smaller
molecular weights and significantly lower proportions of
cross-linked components were observed for PSC, compared
to those of ASC.
Based on the typical band distribution patterns and the
corresponding electrophoretic mobility, it can be suggested
that collagens from the three tissues of grass carp were
mainly composed of type I collagen. This result is in agree-
ment with the previous studies for skin collagen from bigeye
snapper (Benjakul et al., 2010), and skin, scale and bone
collagens from deep-sea redfish (Wang et al. 2008a) and carp
(Duan, Zhang, Du, Yao, & Konno, 2009).
3.2. Amino acid compositions
Table 1 showed the typical amino acid profiles of type I
collagen for ASC and PSC from the scales, skins and swim-
bladders of grass carp. For all collagens, Gly accounted for
nearly one-third of the total amino acids, and Ala, Pro and
Glu/Gln were also found in high amounts in a descending
order, while a negligible amount of Cys and Try were
detected. In addition, all collagens contained Hyp and Hyl,
the unique amino acids identified in collagens. Except for the
first 10 or so amino acid residues at the C-terminus and the
71
last 14 or so at the N-terminus, the normal occurrence of Gly
at every third residue in the collagen is critical for the
formation of the superhelical structure (Bae et al., 2008).
Gly, as the smallest amino acid residue with just a hydrogen
atom side chain, can fit into the center of the superhelix
without any steric hindrance, thus, allowing the three helical
α-chains to pack tightly together to form the final superhelix
with a hydrophobic core (Regenstein & Zhou, 2007).
The total imino acid (Pro and Hyp) contents of both ASC
and PSC from all three tissues of grass carp (157–181 imino
acid residues per 1000 total residues) were lower than those
of pig skin collagen (about 220 imino acid residues per 1000
total residues) and calf skin collagen (about 215 imino acid
residues per 1000 total residues) (Zhang et al., 2007). The
imino acids, especially Hyp, partly contribute to the thermo-
stability of collagens, which is one of the most important
characteristics determining their potential use (Regenstein &
Zhou, 2007). The superhelical structure of a collagen is
maintained by the conformational restrictions imposed by
the pyrrolidine rings of the imino acids, and is further
strengthened by the interchain hydrogen bonds formed with
the hydroxyl group of Hyp (Nagai, Suzuki, & Nagashima,
2008). In general, the collagens from hot- and warm-water
fish have higher total imino acid contents and Hyp contents
than those from cold- and ice-water fish. Grass carp inhabits
the large rivers and lakes in Asia, and the total imino acid
content and Hyp content of collagen from this fish are
comparable to those of collagens produced from other
warm-water fish (Regenstein & Zhou, 2007).
3.3. Peptide hydrolysis patterns
Fig. 2 shows the peptide hydrolysis patterns of ASC and PSC
from the skins, swim-bladders and scales of grass carp
digested using the V8 protease. After the digestion, the band
intensity of the α-chains and crosslinked components
decreased with a concomitant generation of low molecular
weight peptide fragments. In the high molecular weight
regions (4150 kDa), the crosslinked components almost
entirely disappeared for PSC, while some diffused bands were
still observed for ASC. In the low molecular weight regions
(o75 kDa), more small peptide fragments were observed for
PSC, compared to those for ASC. These results suggested that
the limited removal of the telopeptide by pepsin made
collagens from grass carp more susceptible to hydrolysis by
V8 protease. The V8 protease is highly specific for Glu and
Asp residues of proteins and peptides, and the peptide
hydrolysis patterns of a protein is determined by its primary
structure (Jongjareonrak et al., 2005). In general, both ASC
and PSC from the three tissues showed almost similar
peptide hydrolysis patterns, suggesting that the amino acid
sequences of collagens from the three tissues of grass carp
were almost similar.
3.4. Thermal stability
Table 2 shows the Tmax of ASC and PSC from the scales, skins
and swim-bladders of grass carp rehydrated in 0.05 M acetic
acid. A slight variation in Tmax values was observed between
ASC and PSC from each of the three tissues, suggesting that
72 Food Bioscience 9 (2015) 68 –74

Table 1 – Amino acid compositions of ASC and PSC from the scales, skins and swim-bladders of grass carp (residues/1000
total amino acid residues).

Amino acid ASC PSC

Scale Skin Swim bladder Scale Skin Swim bladder

Asp/Asn 49 49 49 48 50 50
Glu/Gln 79 78 84 77 77 80
Ser 37 37 34 36 37 34
His 5 5 6 4 4 4
Gly 343 334 334 342 340 340
Thr 26 25 29 25 26 30
Arg 54 53 55 55 55 55
Ala 127 119 129 125 125 128
Tyr 3 3 3 3 3 3
Cys 0 0 0 0 1 1
Val 20 22 21 22 22 23
Met 14 14 15 14 14 14
Phe 14 15 16 14 14 15
Ile 11 12 12 11 11 12
Leu 21 22 20 21 21 21
Lys 28 29 31 28 29 28
Pro 109 113 88 96 100 97
Hyl 4 2 3 5 4 5
Hyp 57 68 73 73 66 60
Try 0 0 0 0 0 0
Imino acids 166 181 161 169 166 157
Total 1000 1000 1000 1000 1000 1000

Table 2 – Maximum transition temperature (Tmax, 1C) of

ASC and PSC from the scales, skins and swim-bladders of
grass carp rehydrated in 0.05 M acetic acid.

Tissue Tmax of ASC Tmax of PSC

Scale 34.970.0Bc 35.4 70.1Ac

Skin 36.470.1Ab 35.770.1Bb
Swim-bladder 38.370.1Aa 38.070.0Ba

a–c
Means in a column followed by different lower-case letters
differ significantly (po0.05).
A–B
Means in a row followed by different upper-case letters differ
significantly (po0.05).

Fig. 2 – Peptide hydrolysis patterns of ASC (1, 3 and 5) and
PSC (2, 4 and 6) after V8 protease digestion from the skins
(1 and 2), swim-bladders (3 and 4) and scales (5 and 6) of
grass carp.
the triple superhelical structure was still predominant in the
collagen molecule after the limited digestion by pepsin. Both
ASC and PSC from the swim-bladders showed slightly higher
Tmax values than those from the scales and skins (po0.05).
For bighead carp, Liu et al. (2012) also reported that the Tmax
values of PSC from the internal tissues (e.g., swim-bladders
and bones) were slightly higher than those of PSC from the
external tissues (e.g., fins, scales and skins).
The Tmax values of collagens from the three tissues of
grass carp were lower than that of calf skin collagen (40.8 1C)
(Duan et al., 2009), but higher than that of collagen from the
skins of arabesque greenling (15.4 1C) (Nalinanon, Benjakul, &
Kishimura, 2010), the skins of hake (10 1C) (Ciarloa, Paredi, &
Fraga, 1997) and the skins of dusky spinefoot (28.7 1C) (Bae
et al., 2008). Generally, collagens from hot- and warm-water
fish have a higher thermostability than those from cold- and
ice-water fish, but a lower thermostability than those from
land-based animal (Regenstein & Zhou, 2007). Many fish
collagens have Tmax values lower than 30 1C, and fish col-
lagens with Tmax values closer to those of mammalian
collagens may have a larger potential for application in the
food industry (Bae et al., 2008). The comparatively high Tmax
values of collagens from grass carp suggested that they might
be used as potential substitutes for mammalian collagens.
In previous studies, the denaturation temperatures of
scale collagen (Li et al., 2008) and skin collagen (Zhang
et al., 2007) from grass carp were separately reported to be
32.0 and 24.6 1C, respectively. Such variations might partly
result from the differences in the preparation and analysis
 Food Bioscience 9 (2015) 68 –74
methods used in those independent studies. Using the same
extraction procedures and the same analysis method, the
present study showed that the denaturation temperatures of
collagens from the scales, skins and swim-bladders of grass
carp only varied slightly in the range of 34.9–38.3 1C.
4. Conclusion
Both ASC and PSC from the scales, skins and swim-bladders
of grass carp were mainly characterized as type I collagen,
with generally similar electrophoretic patterns, amino acid
profiles and peptide hydrolysis patterns. The limited removal
of the telopeptide by pepsin made collagens more susceptible
to hydrolysis by V8 protease. The thermostability of both ASC
and PSC from the internal tissues (swim-bladders) was only
slightly higher than that of ASC and PSC from the external
tissues (scales and skins). These results suggested that
collagens from the three tissues of grass carp had almost
similar biochemical properties, and could be commercially
used as potential substitutes for mammalian collagens due to
their comparatively high thermostability.
Acknowledgments
This research was partly supported by the National Natural
Science Foundation of China (30901123), the 111 project
(B07029) and the Program for New Century Excellent Talents
in University (NCET-11–0666).
r e f e r e n c e s
Ahmad, M., & Benjakul, S. (2010). Extraction and characterisation
of pepsin-solubilised collagen from the skin of unicorn
leatherjacket (Aluterus monocerous). Food Chemistry, 120,
817–824.
Bae, I., Osatomi, K., Yoshida, A., Osako, K., Yamaguchi, A., & Hara, K.
(2008). Biochemical Properties of acid-soluble collagens
extracted from the skins of underutilised fishes. Food Chemistry,
108, 49–54.
Benjakul, S., Thiansilakul, Y., Visessanguan, W., Roytrakul, S.,
Kishimura, H., Prodprand, T., et al. (2010). Extraction and
characterisation of pepsin-solubilised collagens from the skin
of bigeye snapper (Priacanthus tayenus and Priacanthus
macracanthus). Journal of the Science of Food and Agriculture, 90,
132–138.
Ciarloa, A. S., Paredi, M. E., & Fraga, A. N. (1997). Isolation of
soluble collagen from hake skin (Merluccius hubbsi). Journal of
Aquatic Food Product Technology, 6, 65–77.
Duan, R., Zhang, J. J., Du, X. Q., Yao, X. C., & Konno, K. (2009).
Properties of collagen from skin, scale and bone of carp
(Cyprinus carpio). Food Chemistry, 112, 702–706.
Gelse, K., Pöschl, E., & Aigner, T. (2003). Collagens-structure,
function, and biosynthesis. Advanced Drug Delivery Reviews, 55,
1531–1546.
Heu, M. S., Lee, J. H., Kim, H. J., Jee, S. J., Lee, J. S., Jeon, Y. J., et al.
(2010). Characterization of acid- and pepsin-soluble collagens
from flatfish skin. Food Science and Biotechnology, 19, 27–33.
Jongjareonrak, A., Benjakul, S., Visessanguan, W., Nagai, T., &
Tanaka, M. (2005). Isolation and characterisation of acid and
pepsin-solubilised collagens from the skin of Brownstripe red
snapper (Lutjanus vitta). Food Chemistry, 93, 475–484.
73
Kittiphattanabawon, P., Benjakul, S., Visessanguan, W., Nagai, T.,
& Tanaka, M. (2005). Characterization of acid-soluble collagen
from skin and bone of Bigeye Snapper. Food Chemistry, 89,
363–372.
Kittiphattanabawon, P., Benjakul, S., Visessanguan, W., & Shahidi, F.
(2010). Isolation and properties of acid- and pepsin-soluble
collagen from the skin of blacktip shark (Carcharhinus limbatus).
European Food Research and Technology, 230, 475–483.
Laemmli, U. K. (1970). Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature, 22, 680–685.
Li, C. M., Zhong, Z. H., Wan, Q. H., Zhao, H. L., Gu, H. F., & Xiong, S. B.
(2008). Preparation and thermal stability of collagen from scales
of grass carp (Ctenopharyngodon idellus). European Food Research
and Technology, 227, 1467–1473.
Li, Z. R., Wang, B., Chi, C. F., Zhang, Q. H., Gong, Y. D., Tang, J. J., et al.,
(2013). Isolation and characterization of acid soluble collagens
and pepsin soluble collagens from the skin and bone of Spanish
mackerel (Scomberomorous niphonius). Food Hydrocolloids, 31,
103–113.
Liu, D. S., Liang, L., Regenstein, J. M., & Zhou, P. (2012). Extraction
and characterisation of pepsin-solubilised collagen from fins,
scales, skins, bones and swim bladders of bighead carp
(Hypophthalmichthys nobilis). Food Chemistry, 133, 1441–1448.
Liu, D. S., Regenstein, J. M., Mulvaney, S. J., Boran, G., & Zhou, P.
(2013). Critical review of key analytical issues with gelatins. In
G. Boran (Ed.), Gelatin: Production, Applications and Health
Implications (pp. 31–48). New York: Nova Science Publishers.
Liu, W. T., Li, G. Y., Miao, Y. Q., & Wu, X. H. (2009). Preparation and
characterization of pepsin-solubilized type I collagen from the
scales of snakehead (Ophiocephalus argus). Journal of Food
Biochemistry, 33, 20–37.
Matmaroh, K., Benjakul, S., Prodpran, T., Encarnacion, A. B., &
Kishimura, H. (2011). Characteristics of acid soluble collagen
from scale of spotted golden goatfish (Parupeneus
heptacanthus). Food Chemistry, 129, 1179–1186.
McCormick, R. J. (2009). Collagen. In M. Du, & R. J. McCormick
(Eds.), Applied Muscle Biology and Meat Science (pp. 127–146).
Florida: CRC Press.
Muyonga, J. H., Cole, C. G. B., & Duodu, K. G. (2004).
Characterisation of acid soluble collagen from skins of young
and adult Nile perch (Lates niloticus). Food Chemistry, 85, 81–89.
Nagai, T., Suzuki, N., & Nagashima, T. (2008). Collagen from
common minke whale (Balaenoptera acutorostrata) unesu. Food
Chemistry, 111, 296–301.
Nalinanon, S., Benjakul, S., & Kishimura, H. (2010). Collagens
from the skin of arabesque greenling (Pleurogrammus azonus)
solubilized with the aid of acetic acid and pepsin from
albacore tuna (Thunnus alalunga) stomach. Journal of the Science
of Food and Agriculture, 90, 1492–1500.
Niu, L. H., Zhou, X., Yuan, C. Q., Bai, Y., Lai, K. Q., Yang, F. Y., et al.
(2013). Characterization of tilapia (Oreochromis niloticus) skin
gelatin extracted with alkaline and different acid
pretreatments. Food Hydrocolloids, 33, 336–341.
Regenstein, J. M., Chaudry, M. M., & Regenstein, C. E. (2003).
The Kosher and Halal food laws. Comprehensive Reviews in Food
Science and Food Safety, 2, 111–117.
Regenstein, J. M., & Zhou, P. (2007). Collagen and gelatin from
marine by-product. In F. Shahidi (Ed.), Maximising the Value of
Marine By-products (pp. 279–303). Florida: CRC Press.
Rodziewicz-Motowidło, S., Śladewska, A., Mulkiewicz, E.,
Kołodziejczyk, A., Aleksandrowicz, A., Miszkiewicz, J., et al.
(2008). Isolation and characterization of a thermally stable
collagen preparation from the outer skin of the silver carp
Hypophthalmichthys molitrix. Aquaculture, 285, 130–134.
Saito, M., Kunisaki, N., Urano, N., & Kimura, S. (2002). Collagen as
the major edible component of sea cucumber (Stichopus
japonicus). Journal of Food Science, 67, 1319–1322.
74 Food Bioscience 9 (2015) 68 –74
Wang, L., An, X. X., Yang, F. M., Xin, Z. H., Zhao, L. Y., & Hu, Q. H.
(2008a). Isolation and characterisation of collagens from the
skin, scale and bone of deep-sea redfish (Sebastes mentella).
Food Chemistry, 108, 616–623.
Wang, L. Z., Yang, B., Wang, R., & Du, X. Q. (2008b). Extraction of
pepsin-soluble collagen from grass carp (Ctenopharyngodon
idella) skin using an artificial neural network. Food Chemistry,
111, 683–685.
Yang, H. S., Wang, Y. F., Jiang, M. K., Oh, J. H., Herring, J., & Zhou, P.
(2007). 2-Step optimization of the extraction and subsequent
physical properties of channel catfish (Ictalurus punctatus) skin
gelatin. Journal of Food Science, 72, C188–C195.
Zague, V. (2008). A new view concerning the effects of collagen
hydrolysate intake on skin properties. Archives of
Dermatological Research, 300, 479–483.
Zhang, J. J., Duan, R., Ye, C., & Konno, K. (2010). Isolation and
characterization of collagens from scale of silver carp
(Hypophthalmichthys molitrix). Journal of Food Biochemistry, 34,
1343–1354.
Zhang, Y., Liu, W. T., Li, G. Y., Shi, B., Miao, Y. Q., & Wu, X. H.
(2007). Isolation and characterization of pepsin soluble
collagen from the skin of grass carp (Ctenopharyngodon idella).
Food Chemistry, 103, 906–912.