Professional Documents
Culture Documents
PII: S0141-8130(16)30356-7
DOI: http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.04.048
Reference: BIOMAC 6016
Please cite this article as: Ya’nan Yang, Caiyan Li, Wei Song, Wei Wang, Guoying
Qian, Purification, optimization and physicochemical properties of collagen from
soft-shelled turtle calipash, International Journal of Biological Macromolecules
http://dx.doi.org/10.1016/j.ijbiomac.2016.04.048
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.
Purification, optimization and physicochemical properties of
*Corresponding authors
Dr. Caiyan Li will handle correspondence at all stages of refereeing and publication,
also post-publication.
Tel./fax: +86-574-88222991
1
Abstract
The present work was to optimize the purification conditions for soft-shelled
Single-factor test and orthogonal method L9 (34) were employed with the STCC
recovery yield as indicator. The optimum purification conditions were obtained when
scanning, FTIR, solubility, thermal behavior and amino acid analysis. The results
showed that STCC contained high hydroxyproline content than that of other fishery
skins, belonging to typical type I collagen in form of [α1(I)] 2α2(I). FTIR spectra of
STCC were quite similar to other aquatic animals’ collagens. It has the lowest
solubility dropped. The denaturation temperature (Td) and melting temperature (Tm)
were 35.1 oC and 105.14 oC, respectively. Morphology of STCC depicted as regular
and porous network structure by SEM. In general, the results suggested that turtle
physicochemical properties
2
1. Introduction
3
reported the extraction and characterization of collagen from turtle skin [13], lung [14]
and calipash [15, 16]. And optimum extraction conditions were also obtained. The
present study was further attempted to determine the purification conditions for
extracted soft-shelled turtle calipash collagen (STCC) and to characterize the collagen
by several physicochemical methods in order to discuss the potential of STCC in
biomaterial application area.
The soft-shelled turtles were provided by the turtle farm in Ningbo, China (body
weight of 500±50 g). They were put in the nylon mesh bag under normal temperature
when transported to our laboratory. The calipash tissues were dissected under iced
conditions, cut into small pieces and stored at -20C for further use. All experimental
procedures were approved by the Animal Ethics Committee of Zhejiang Wanli
University. The chemical reagents including pepsin (3000 U/g) and L-hydroxyproline
standard were purchased from Sangon Biotech (Shanghai) Co. Ltd. All reagents used
in this study were analytical grade.
The collagen was extracted by pepsin from soft-shelled turtle calipash. The
tissues were pretreated in 0.1 M NaOH for 24 h at 4oC to remove non-collagenous
proteins and then with 10% isopropyl alcohol to remove fat. Collagen was crudely
extracted from calipash in 25 volumes (v/w) of 0.5M acetic acid containing 2mg/mL
pepsin for 24h with continuous stirring. Supernatant was collected by centrifugation
and then the crude collagen solution was salted-out by the addition of NaCl.
The salted out STCC precipitate was further obtained by centrifugation at
10,000g for 30 min. As for the calculation of recovery yield of STCC in salting out
technology optimization, the precipitate could be used directly for collagen content
determination without the remove of salt. But for the STCC characterization, the
pellets were dissolved in 0.5 M acetic acid and the resulting solution was dialyzed
4
against distilled water for 24 h under 4 oC to remove salt. The resulting dialysates was
lyophilized for further use [19].
Effects of three factors including salting out time, STCC concentration and NaCl
concentration were investigated on STCC recovery yield, respectively. The design of
single factor experiment was shown in Table 1. In order to avoid the denaturation of
collagen solution and ensure the purity and yield of collagen, according to the
previous references [20] and our preliminary experiment, the fixed levels of salting
out time, NaCl concentration and STCC concentration were selected at 24 h, 3 M, 10
g/L respectively. One factor was changed from the lowest level to the highest, while
the other two factors were fixed at one level.
The recovery yield of STCC was calculated and expressed as collagen content in
salted out solution / crude solution of STCC. The collagen content was determined by
measuring the content of hydroxyproline, which is the characteristic amino acid in
collagen, by using spectrophotometric analyzer (NanoDrop2000, Thermo Fisher
Scientific, USA). The procedures were described in our previous study [14].
On the basis of the single-factor test described in 2.3.1, an orthogonal L9 (34) test
design with four factors and three levels was used to investigate the optimal salting
out condition of STCC. As seen from Table 2, nine groups of salting out experiment
were carried out at salting out time (A) 12, 24, 36 h, NaCl concentration (B) 2, 3, 4 M,
STCC concentration (C) 6, 8, 10 g/L. The STCC recovery yield (%) was the
dependent variable, and the data of the orthogonal experiment was analyzed by SPSS
19.0 software.
5
(PowerpacBasic, Bio-rad, USA). After electrophoresis, the gel was stained with 0.1%
(w/v) Coomassie blue R-250 in 15% (v/v) methanol and 10% (v/v) acetic acid for 30
min and destained with 30% (v/v) ethanol and 10% (v/v) acetic acid. High molecular
weight markers were used to estimate the molecular weight of proteins. The
electrophoresis pattern was imaged by ultraviolet spectrophotometer (Gel Doc™ XR+,
Bio-rad, USA). The crude and purified collagens were both used for SDS-PAGE to
compare the purification effects and the composition.
2.5. UV scanning
The ultraviolet absorption spectra of the purified collagen samples were scanned
by UV spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, USA) between
190 and 400 nm with an interval of 1 nm .
STCC samples and calipash of Pelodiscus Sinensis were both hydrolyzed under
standard conditions. The amino acid composition was analyzed by an Amino Acid
Auto Analyzer (L-8900, Hitachi Ltd., Japan). The amino acid content was expressed
as the percentage of individual amino acid by the total content.
6
The final volume was adjusted to 10 mL with distilled water. The mixture was stirred
and centrifuged at 10,000 g for 30 min. Protein concentration of the supernatant was
measured by the Lowry method [22] using bovine serum albumin as a standard.
Relative solubility was calculated by dividing the highest solubility value by the
solubility of the test sample.
In the determination of STCC solubility at different NaCl concentration, 5 mL of
6 mg/mL STCC solution was mixed with six groups of 5 mL NaCl solution (0%, 2%,
4%, 6%, 8%, 10% and 12% (w/v) ), separately, to obtain a series of STCC
concentrations (0%, 1%, 2%, 3%, 4%, 5% and 6% (w/v) ). The protein content and
collagen solubility was measured as described above.
The melting temperature (Tm) was defined as the point at which the physical
form of collagen was transformed from solid to liquid. The primary structure
(sequence of amino acid) of collagen was destroyed when the temperature was higher
than the Tm. The melting temperature of collagen was assessed with DSC (Sataram,
DSC-30, France). The STCC was smashed to powder before measuring. The pan was
heated at the rate of 10 oC /min from 20 oC to 140 oC with an empty pan as reference.
The Tm of the collagens was defined as the temperature of endothermic peak [23].
8
Effects of salting out time, NaCl concentration and STCC concentration on
recovery yield of STCC were seen in Fig. 1(A), (B) and (C), separately. The Fig. 1(A)
showed that there was a continuous increasing of salting out time when it was ranging
from 1 h to 24 h and the highest recovery yield of STCC was observed when the time
was 24 h. Then a sharp drop at 36 h was observed. As showed in the Fig. 1(B), the
recovery yield of STCC displayed an increasing trend with the NaCl concentration
increasing from 1 M to 3 M, reached the peak value at 3 M and decreased after 3 M.
This was in accordance with the results of bovine collagen, of which the speed of
protein salting out was enhanced as the NaCl concentration increased [20].
Moreover, as the Fig. 1(C) exhibited, the recovery yield of STCC increased
continuously when the STCC concentration was in the range of 2 g/L to 8 g/L and
rose to the highest value at 8 g/L, then declined at 10 g/L. It is probably because when
the collagen concentration was higher than 8 g/L, the solution became saturated and it
was difficult for NaCl to dissolve and thus the yield decreased.
The investigated levels of each factor were selected depending on the above
experimental results of the single-factor and examined using an orthogonal test design
L9 (34). Independent variables with three optimal levels, salting out time (12, 24, 36 h),
NaCl concentration (2, 3, 4 M), STCC concentration (6, 8, 10 g/L) are listed in Table
2, and the orthogonal test results and extreme difference analysis are also presented in
Table 2, with the recovery yield (%) of STCC as dependent variable.
As seen from the results of Table 2, the highest recovery yield (%) of STCC
(80.47%) was obtained when salting out time, NaCl concentration and STCC
concentration were A2B1C2 (24 h, 2 M, 8 g/L). According to the R values, the
influences to the mean extraction yield decrease in the order of B>C>A. The yield of
collagen was significantly influenced by NaCl concentration. However, we cannot
directly choose the corresponding salting out conditions as the best technology. In
addition, three groups of parallel experiments were operated under the optimum
condition A2B1C2 (24 h, 2 M, 8 g/L) to verify if it was feasible to get the highest
9
recovery yield of STCC, the average value of the verification test was 81.35%, which
demonstrated that the optimum condition A2B1C2 can get the highest recovery yield of
STCC.
There are few reports about effects of salting out on the collagen purification. A
method of NaCl salting out was also conducted in bovine lung tube by Wang (2012)
[20], in which the recovery yield of bovine type II collagen was as high as 90.32%
under the optimum salting out conditions. The reasons of collagen yield differences
could be the different collagen sources as well as the type. It is generally recognized
that the solubility of different types of collagen may vary with NaCl concentrations,
which needs further explanation by collagen characterization.
The SDS-PAGE pattern of crude and purified collagens was shown in Fig. 2,
which demonstrated that the salted out collagen has high degree of purification. And
the SDS-PAGE patterns also indicated that the STCC was composed of two α1 chains
and one α2 chain, in the typical I form of [α1(I)]2α2(I), which was not only similar to
collagen of soft-shelled turtle skin [13] but also typical I collagens of many other
aquatic tissues such as balloon fish skin [7], sea cucumber skin [9], blacktip shark
skin [10], spanish mackerel bone [12], Amur sturgeon skin [23], skin, bone and
muscle of leather jacket [26] and pipefish skin [27], etc, which illustrated there were
no differences among the subunit composition of typical I collagens from different
aquatic tissues. And the dimers (β-chains) and trimers (γ-chains) of α chain were seen
in the electrophoresis pattern, which proved the triple helix of STCC was not
destructed in the process of extraction and purification [27].
It was found that the molecular weight of α1 and α2 was about 130 kDa and 120
kDa, respectively, thus the molecular weight of STCC was about 380 kDa, which was
significantly higher than that of collagens from some aquatic tissues mentioned above
such as unicorn leatherjacket skin (about 340 kDa) [21], pipefish skin (about 280 kDa)
[27] and squid skin (about 280 kDa) [28]. It showed that while the subunit
composition of typical I collagens from different aquatic tissues was same with each
10
other, the molecular weight differences still existed among them. Duan et al. [29]
stated that the molecular weight of collagen had some relations with the thermal
stability and the collagen with higher molecular weight may have greater thermal
stability.
UV scanning can be used to analyze the collagen, because triple helical collagen
has maximum absorbance peak at about 230 nm due to the groups C=O, –COOH,
CONH2 in polypeptides chains of collagen [30, 31]. The UV absorption spectrum of
STCC at the wavelengths between 190 and 400 nm were presented in Fig. 3. It can be
seen that STCC had a sharp and strong absorption peak at 230 nm and did not have
typical maximum ultraviolet absorption peak at 280 nm as most other types of
proteins. The reasons might be that STCC contained very low or even no content of
tryptophan, tyrosine and phenylalanine, which has the typical absorption peak at 280
nm. The results were in accordance with that of collagens from other aquatic animals
reported [25] and also indicate the efficient removal of non-collagenous protein in the
process of collagen extraction.
11
involved with triple helical structure of collagen [21]. Amide III band of STCC was
found at 1214 cm-1, indicating that pepsin hydrolysis and salting out had no
significant effect on the triple helix structure of STCC [32]. FTIR spectra of STCC
were quite similar to that of to collagen from horse-faced fish skin, of which the
amides A, B, I, II, and III were 3423, 2939, 1655, 1552 and 1238 cm-1, respectively
[33]. The results were also similar to other aquatic collagen sources which include
sailfish [24], albacore tuna [21], and yellowfin tuna [32]. Lower wavenumber of
amides A (3294 and 3333 cm-1 ) in the collagen from bovine and porcine skin were
also reported [21, 32]. These indicated more NH group in mammalian collagen was
involved in hydrogen bond [23].
The amino acid compositions of turtle calipash and STCC were presented in
Table 3, respectively. The calipash and STCC both had glycine (23.15% and 19.99%)
as their major amino acid, followed by alanine (12.76% 11.38%), proline (10.27% and
12.15%), glutamic acid (11.51% and 11.51%) and the hydroxyproline (8.22% and
10.84%) contents, respectively. In general, glycine is the most abundant amino acid in
collagen, and the amount was approximately 33%. However, according to some
references, it depends on the animal species and varied from 13% to 40% [24, 30].
Glycine was generally occurs uniformly, at every third residue throughout most of the
collagen molecules, except for the first 14 amino acid from the N-terminus and the
first 10 from the C-terminus. The data showed that the contents of proline and
hydroxyproline in STCC were higher than that in calipash after extraction and
purification. And as reported by Nagai [13] the content of hydroxyproline in collagen
would affect the thermal stability with positive correlation, hydroxyproline of STCC
was higher than that of unicorn leather jacket skin (8.3%) [21], Amur sturgeon skin
(10.33%) [23] and yellowfin tuna skin (8.0%) [34].
The contents of tryptophan, tyrosine and phenylalanine in STCC were 0.01%,
0.00% and 0.47%, respectively, revealing that the content of aromatic residues in
STCC was very low, which was consistent with the UV absorption spectrums of
12
STCC (Fig. 3). In contrast, the content of these three aromatic residues in calipash
were 0.02%, 2.45% and 0.98%, respectively. Being the typical amino acid of
non-collagenous protein, the reduced contents of aromatic residues in STCC was
therefore suggested that it was of high purity.
The effect of pH on the solubility of STCC was shown in Fig. 5(A). It can be
seen that the solubility of STCC had an upward trend when pH ranged from 1 to 3,
revealing that the strong acid may lead to the denaturation of STCC. And then a sharp
decrease in solubility was observed in the pH range of 3 to 6. The lowest solubility
point of pH 6 was therefore defined as the isoelectric point (pI) of STCC. The
dissolved protein was found to precipitate in this pH range probably due to the
hydrophobic interaction among collagen molecules, which increase at pI value [21,
34]. Protein aggregation and precipitation are induced by the pI of almost zero total
charge of the protein molecules [21, 32]. Slight increase in solubility was noticeable
in an alkaline pH from 7 and 10, which could be explained by the repulsive effect of
collagen molecules.
In Fig. 5(B), solubility of STCC was not influenced when NaCl concentration
was lower than 2% (w/v), and it had a sharp decrease when NaCl concentration was
from 2% to 6% (w/v). Afterwards it decreased slowly when NaCl concentration was
from 8% to 12% (w/v), thus the minimum solubility of STCC was at 12% NaCl
concentration. Solubilities of other aquatic collagens at different NaCl concentration
have been reported [21, 34, 35]. Consistent decreased solubility behavior at high NaCl
concentration can be attributed to the salting-out effect. The solubility declined by
enhancing hydrophobic sites interactions between protein chains, leading to protein
precipitation.
Overall, STCC generally showed higher solubility in acidic conditions and lower
solubility at high NaCl concentration, which is related to the collagen with different
characteristic and molecular properties [21, 36].
The SEM images of STCC were depicted in Fig. 7. Surface (A) and cross-section
(B) of STCC sponge were both observed under 30, 100 and 300 magnifications,
14
respectively. It could be seen in the Fig. 7 that the purified STCC was presented as
purely white sponge, and the SEM microscopic structure of STCC showed a
homogenous, multi-layered aggregated structure. The surface and cross-section of
collagen scanned by the SEM under 30 magnifications were presented as loose,
porous and fibrous structure, and the pores were orderly sequenced (Fig. 7 A2, B2).
The SEM microstructure was observed as high porosity and large aperture both under
100 and 300 magnifications (Fig. 7 A3, A4, B3, B4). In general, the surface SEM image
appeared to be spongy like structures with irregularly distributed and interconnected
pores (Fig. 7 A2-A4), whereas the cross-section image indicated intersecting fibers
with parallel orientation and meshwork appearance, regular-shaped like strips (Fig. 7
B2-B4).
Scaffold microstructure, such as pore size and porosity, is widely recognized as
important parameters for biomaterial evaluation [24]. Recently, the collagen has been
focused by the biomaterial areas because of its excellent characteristics, in which the
three-dimensional structure of collagen was of great importance, for it can not only
provide plenty of space for cell growth [39], but also could be used as drug carrier. In
this study, it was the first time that this kind of collagen was investigated by SEM and
the morphologies suitable for biomedical engineering was just found. The loose,
porous and fibrous structure of STCC scanned by the SEM would provide powerful
physical basis for its further use in biomaterial areas.
4. Conclusions
In this study, collagen was extracted from the calipash of soft-shelled turtle by
pepsin and the salting out conditions of collagen was also optimized. The
characterization of physical and chemical properties of purified collagen indicated the
feasibility of using the turtle calipash as a good alternative source of mammalian
collagen. Besides, superiorities to other aquatic collagens, i.e. the thermal behavior,
which may have more privilege to be used in biomaterial areas was observed.
However, there are still limits for the direct use of calipash collagen in biomaterial,
15
because cross-linking operation to strengthen mechanical properties as well as the
biocompatibility and toxicity level of collagen should be further investigated.
Acknowledgment
This study was funded by the Research Project of Public Welfare Technology
Application in Zhejiang Province (No. 2014C32072), the Natural Science Foundation
for Young Scientists of Zhejiang Province (No. LQ13C190001) and the Ningbo
Natural Science Foundation (2015A610259). Dr. Qian was financed by the State
Oceanic Administration of China (201405015). Mr. Song was also supported by the
Zhejiang Provincial Top Key Discipline of Biological Engineering (No. ZS2015008).
16
References
[1] K. Takaki, Designed triple-helical peptides as tools for collagen biochemistry and
matrix engineering, Phil. Trans. R. Soc. B 362 (2007) 1281–1291.
[2] L.C. Abraham, E. Zuena, B.P. Ramirez, D.L. Kaplan, Guide to collagen
characterization for biomaterial studies, J. Biomed. Mater. Res. B 87 (2008)
264–285.
[3] B. Daniele, B. Tord, L. Jan, The influence of a biomaterial on the closure of a
marginal hard tissue defect adjacent to implants. An experimental study in the
dog, Clin. Oral Implan. Res. 15 (2008) 285–292.
[4] B.L.E. Castro, J.L. Proffitt, S.Bloor, P.D. Sibbons, Effect of crosslinking on the
performance of a collagen-derived biomaterial as an implant for soft tissue repair:
A rodent model, J. Biomed. Mater. Res. B 95 (2010) 239–249.
[5] K.M. Senthil, K. Shanmugam, S. Ramasamy, S.P. Kumar, Triphala incorporated
collagen sponge--a smart biomaterial for infected dermal wound healing, J. Surg.
Res. 158 (2009) 162–170.
[6] M. Hiram, U. Montoya, J. Luis, Maribel, P. Jatomea, Hisila, Ofelia, J. Luis,
Enrique, J. Marina, E. Brauer, Jumbo squid (Dosidicus gigas) mantle collagen:
Extraction, characterisation, and potential application in the preparation of
chitosan-collagen biomaterial, Bioresour. Technol. 101 (2010) 4212–4219.
[7] Y.R. Huang, C.Y. Shao, H.H. Chen, B.C. Huang, Isolation and characterisation of
acid and pepsin-solubilised collagens from the skin of balloon fish (Diodon
holocanthus), Food Hydrocolloid. 25 (2011) 1507–1513.
[8] M.C. Gómez-Guillén, B. Giménez, M.E. L. Caballero, M.P. Montero, Functional
and bioactive properties of collagen and gelatin from alternative sources : A
review, Food Hydrocolloid. 25 (2011) 1813–1827.
[9] N. Adibzadeh, S. Aminzadeh, S. Jamili, A.A. Karkhane, N. Farrokhi, Appl,
Purification and characterization of pepsin-solubilized collagen from skin of sea
cucumber Holothuria parva, Biochem. Biotechnol. 173 (2014) 143–154.
[10] P. Kittiphattanabawon, S. Benjakul, W. Visessanguan, F. Shahidi, Isolation and
17
properties of acid- and pepsin-soluble collagen from the skin of blacktip shark
(Carcharhinus limbatus), Eur. Food Res. Technol. 230 (2010) 475–483
[11] H.S. Jeong, J. Venkatesan, S.K. Kim, Isolation and characterization of collagen
from marine fish (Thunnus obesus), Biotechnol. Bioproc E. 18 (2013)
1185–1191.
[12] J. Kozlowska, A. Sionkowska, J. Skopinska-Wisniewska, K. Piechowicz,
Northern pike (Esox lucius) collagen: Extraction, characterization and potential
application, Int. J. Biol. Macromol. 81 (2015) 220–227.
[13] N. Nagai, H. Kobayashi, S. Katayama, M. Munekata, J. Biomater, Preparation
and characterization of collagen from soft-shelled turtle (Pelodiscus sinensis)
skin for biomaterial applications, J. Biomat. Sci-Polym. E. 20 (2009) 567–576.
[14] W. Song, W. Chen, Y.N. Yang, C.Y. Li, G.Y. Qian, Extraction optimization and
characterization of collagen from the lung of soft-shelled turtle Pelodiscus
sinensis, Int. J. Sci. Nutr. Food 3 (2014) 270–278.
[15] J.F. Lu, Q. Wan, Z.M. Yin, L. Lin, S.B. Weng, Y.W. Ye, S.T. Jiang, Extraction and
characterization of collagen from calipash of Chinese soft-shelled turtle
(Pelodiscus sinensis), J. Fisheries China 34(2010) 981-988.
[16] Y.T. Tian, H.H. Shen, W. Song, Y.T. Mao, Y.R. Zhou, X. Peng, C.Y. Li, G.Y. Qian,
Extraction technical optimization and characterization of collagen from Chinese
soft-shelled turtle (Pelodiscus sinensis), JiangSu Agric. Sci. 41(2013)265-269.
[17] C.C. Liu, Y. Liu, Y.Z. Jin, Z.P. Ding, Y.S. Li, J.L. Li, Extraction and antioxidant
activity of collagen from the Chinese soft-shelled turtle (Pelodiscus sinensis),
Adv. Mater. Res. 152-153(2011)1788-1792.
[18] FAO. Cultured Aquatic Species Information Programme Trionyx sinensis
(Weigmann, 1834) [DB/OL]. http://www.fao.org/fishery/culturedspecies/
Trionyx_sinensis/en
[19] P. Roberta, Ronaldo, N.M. Pitombo, Care during freeze-drying of bovine
pericardium tissue to be used as a biomaterial: A comparative study, Cryobiology.
63 (2011) 61–66.
[20] C.H. Wang, Extraction, purification and identification of collagen from bovine
18
lung tube. Master's Thesis. Jilin Agric. Univ. 2012. p 22-23.
[21] M. Ahmad, S. Benjakul, Extraction and characterisation of pepsin-solubilised
collagen from the skin of unicorn leatherjacket (Aluterus monocerous), Food
Chem. 120 (2011) 817–824.
[22] O.H. Lowry, N.J.Rosebrough, A.L.Farr, R.J.Randall, Protein measurement with
Folin phenol reagent, J. Biol. Chem. 193 (1951) 256–275.
[23] L. Wang, Q.F. Liang, T.T. Chen, Z.B. Wang, J.M. Xu, H.L. Ma, Characterization
of collagen from the skin of Amur sturgeon (Acipenserschrenckii), Food
Hydrocolloid. 38 (2014) 104–109.
[24] S. Tamilmozhi, A. Veeruraj, M. Arumugam, Isolation and characterization of acid
and pepsin-solubilized collagen from the skin of sailfish (Istiophorus
platypterus), Food Res. Int. 54 (2013) 1499–1505.
[25] E. Jeevithan, W.H. Wu, N.P. Wang, L. He, B. Bao, Isolation, purification and
characterization of pepsin soluble collagen isolated from silvertip shark
(Carcharhinus albimarginatus) skeletal and head bone, Process Biochem. 49
(2014) 1767–1777.
[26]N. Muralidharan, R.J. Shakila, D. Sukumar, G. Jeyasekaran, Skin, bone and
muscle collagen extraction from the trash fish, leather jacket (Odonus niger) and
their characterization, Int. J. Food Sci. Tech. 50 (2013) 1106–1113.
[27] S.B. Khan, Z.J. Qian, B.M. Ryu, S.K. Kim, Isolation and biochemical
characterization of collagens from seaweed pipefish, Syngnathus schlegeli,
Biotechnol. Bioproc. E. 14 (2013) 436–442.
[28] A.Veeruraja, A.Muthuvel, A.Thangappan, B.Thangavel, Isolation and
characterization of collagen from the outer skin of squid (Doryteuthis
singhalensis), Food Hydrocolloid. 43 (2015) 708–716.
[29] R. Duan, J. Zhang, X. Du, X. Yao, K. Konno, Properties of collagen from skin,
scale and bone of carp (Cyprinus carpio), Food Chem. 112 (2009) 702–706.
[30] Y.K. Lin, D.C. Liu, Comparison of physical-chemical properties of type I
collagen from different species, Food Chem. 99 (2006) 244–251
[31] C.B. Zhao, Y.D. Huang, Y.H. Li. Extraction of collagen from cattle tendons. J.
19
Harbin Inst. Technol. 36 (2004) 515–519.
[32] S. Sankar, S. Sekar, R. Mohan, S. Rani, J. Sundaraseelan, T.P. Sastry, Preparation
and partial characterization of collagen sheet from fish (Lates calcarifer) scales,
Int. J. Biol. Macromol. 42 (2007) 6–9.
[33] S.Q. Cao, S.S. Xia, L.Liu, X.Y.Qi, Extraction and characterization of collagen
from Navodon septentrionalis skin. J. Chin. Inst. Food Sci. Technol.10 (2014)
117-123.
[34] J.W. Woo, S.J. Yu, S.M. Cho, Y.B. Lee, S.B. Kim, Extraction optimization and
properties of collagen from yellow fin tuna (Thunnus albacares) dorsal skin,
Food Hydrocolloid. 22 (2008) 879–887.
[35] L.T. Minh Thuy, E. Okazaki, K. Osako, Isolation and characterization of
acid-soluble collagen from the scales of marine fishes from Japan and Vietnam,
Food Chem. 149 (2014) 264-270.
[36] G.P. Wu, , X.M. Wang, , L.P. Lin, , S.H. Chen, Q.Q. Wu, Isolation and
characterization of pepsin-solubilized collagen from the skin of black carp
(Mylopharyngdon piceus), Adv. Biosci. Biotechnol. 5 (2014) 642–650.
[37] Y. Zhang, W.T. Liu, G.Y. Li, , B. Shi, Y.Q. Miao, X.H. Wu, Isolation and partial
characterization of pepsin-soluble collagen from the skin of grass carp
(Ctenopharyngodon idella), Food Chem. 103 (2006) 906–912.
[38] L.S. Senaratne, P.J. Park, S.K. Kim, Isolation and characterization of collagen
from brown backed toadfish (Lagocephalus gloveri) skin, Bioresour. Technol. 97
(2006) 191–197.
[39]S.T.M. Nillesen, P.J. Geutjes, R. Wismans, J. Schalkwijk, W.F. Daamen, T. H. van
Kuppevelt, Increased angiogenesis and blood vessel maturation in acellular
collagen-heparin scaffolds containing both FGF2 and VEGF, Biomaterials. 28
(2006) 1123-1131.
20
Figure Captions
Fig. 1. Effects of salting out time, NaCl concentration and collagen concentration on
recovery yield of the calipash collagen from Pelodiscus sinensis. A, salting out time;
B, NaCl concentration; C, collagen concentration
Fig. 6. Thermal behavior results of the calipash collagen from Pelodiscus sinensis. A,
thermal denaturation curve; B, melting temperature curve.
21
Fig.1
22
Fig.2
23
Fig.3
24
Fig.4
25
Fig.5
26
Fig.6
27
Fig.7
28
Tables
Table 1 Design of single factor experiment
Levels Factors
Salting out time (h) NaCl concentration (M) collagen concentration (g/L)
1 1 1 2
2 12 2 4
3 24 3 6
4 36 4 8
5 48 5 10
29
Table 2 Design and analysis of L9 (34) orthogonal experiment
30
Table 3 Amino acid composition of turtle calipash and collagen from calipash
Composition (%)
Amino acid
turtle calipash turtle calipash collagen
31