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Accepted Manuscript

Title: Purification, optimization and physicochemical


properties of collagen from soft-shelled turtle calipash

Author: Ya’nan Yang Caiyan Li Wei Song Wei Wang


Guoying Qian

PII: S0141-8130(16)30356-7
DOI: http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.04.048
Reference: BIOMAC 6016

To appear in: International Journal of Biological Macromolecules

Received date: 29-11-2015


Revised date: 14-4-2016
Accepted date: 16-4-2016

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

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Purification, optimization and physicochemical properties of

collagen from soft-shelled turtle calipash


Ya’nan Yang, Caiyan Li, Wei Song, Wei Wang, Guoying Qian
College of Biological and Environmental Sciences, Zhejiang Wanli University,
Ningbo 315100, China

*Corresponding authors

Caiyan Li, Tel./fax: +86-574-88222991, E-mail: licy82@163.com

Guoying Qian, Tel./fax: +86-574-88222298, E-mail: qiangy@zwu.edu.cn

Dr. Caiyan Li will handle correspondence at all stages of refereeing and publication,
also post-publication.

Email: licy82@163.com; licy@zwu.edu.cn.

Tel./fax: +86-574-88222991

Address: College of Biological and Environmental Sciences, Zhejiang Wanli


University, South Qianhu Road 8, Ningbo 315100, P.R.China

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Abstract
The present work was to optimize the purification conditions for soft-shelled

turtle (Pelodiscus sinensis) calipash collagen (STCC) isolated by pepsin and to

explore collagen physicochemical properties for potential biomaterial applications.

Single-factor test and orthogonal method L9 (34) were employed with the STCC

recovery yield as indicator. The optimum purification conditions were obtained when

NaCl concentration, collagen concentration and purification time were 2 M, 8 g/L,

and 24 h, respectively. Purified STCC were characterized by SDS-PAGE, UV

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 at pH 6, and when NaCl concentration decreased from 2% to 6% (w/v),

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

calipash can be exploited as alternatives to mammalian collagen and could also be

used for biomedical applications as a potential new material.

Keywords: Pelodiscus sinensis calipash collagen; purification optimization;

physicochemical properties

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1. Introduction

Collagen is a very important major structural protein supporting connective


tissues, which has a unique triple-helical structure formed by three polypeptide chains
[1]. Collagen possesses many superior features, such as high tensile strength, good
biocompatibility, low antigenicity, low irritation and low cytotoxicity [2], and can be
used in burns, trauma, corneal disease, orthopedic, hard tissue repair [3], soft tissue
repair [4], wound healing [5] and other extensive medical and health purposes,
attributing to its fibril forming ability which is useful in biomaterial applications. The
excellent biological properties have demonstrated its potential as an ideal biomaterial.
However, the improvement of biological properties of collagen molecules is of great
importance in the research and development of novel collagen-based biomaterial.
Despite the advantage, the application of collagen as a promising biomaterial also has
some drawbacks. For instance, the preparation and purification procedures of collagen
are always complicated.
The traditional collagen in biomaterial applications was mainly extracted from
pig, cattle and other terrestrial animals [6]. However, the collagen product from
mammalian animals was restricted due to mad cow disease, foot-and -mouth disease
and other problems [7]. In recent years, aquatic collagen as an alternative has received
increased attention because of more biological safety [8]. Collagens were extracted
from the skin, muscle or bones of various kinds of fishes [9-12]. The soft-shelled
turtles, Pelodiscus sinensis, which is a commercially important and delicious aquatic
species in Asian countries, especially in China, Japan and southern Taiwan area, have
been used for collagen extraction [13-16]. Compared to other coldwater fishes, the
soft-shelled turtle is an aquatic animal living in relatively higher ambient environment
at around 30 °C [13]. The turtle collagens have higher denaturation temperature (Td)
and may have the advantage of higher thermal stability in biomedical applications.
Additionally, the global aquaculture production of Pelodiscus sinensis was up to
347,587 tons in 2013 and approximately more than 140,000 tons were produced from
2005 in China alone [17, 18]. Our colleagues and other researchers have previously

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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.

2. Materials and Methods

2.1. Materials and Chemical Reagents

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 -20C 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.

2.2. Extraction and purification of STCC

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

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against distilled water for 24 h under 4 oC to remove salt. The resulting dialysates was
lyophilized for further use [19].

2.3. Optimization of Salting Out Technology

2.3.1. Single-Factor Test

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].

2.3.2. Orthogonal L9 (34) Test

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.

2.4. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

The electrophoresis was performed by using the discontinuous Tris-HCl/glycine


buffer system with 8% separation gel and 5% stacking gel using a vertical cell
(MiniPROTEAN3, Bio-rad, USA) at voltage of 100v for about 100 min

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(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 .

2.6. Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectra of purified STCC were obtained by Fourier Transform Infrared


Spectrometer (VERTEX 70, Bruker, Germany). One-two mg of collagen samples
were tableted with KBr and then scanned in a range of 400-4000 cm-1 with 32 scans
per sample at a resolution of 4 cm-1 [11].

2.7. Amino-Acid Composition

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.

2.8. Collagen Solubility

Effect of pH and NaCl on collagen solubility was measured according to the


method described in Ahmad et al. [21] with slight modification. Briefly, STCC was
dissolved in 0.5 M acetic acid to obtain a final of 3 mg/mL or 6 mg/mL and the
mixture was stirred at 4 oC until STCC was completely solubilised.
STCC solution (8 mL, 3 mg/mL) was transferred to a series of centrifuge tubes
to obtain pH values ranging from 1.0 to 10.0 by addition of 6 M NaOH or 6 M HCl.

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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.

2.9. Thermal behaviors

2.9.1. Thermal Denaturation Temperature(Td)Relative viscosity

The thermal denaturation temperature (Td) was defined as the temperature at


which the 50% collagen molecules were denatured and lost biological activity, i.e. the
viscosity of collagen was changed for half. The Td of STCC was calculated by
Ubbelohde viscometer in this study. STCC samples were dissolved in 0.1 M acetic
acid and 0.2 M sodium acetate to obtain a concentration of 0.03% (w/v), and then the
Td was calculated with the time of collagen solution flow out the capillary as the
index. The Ubbelohde viscometer (Shanghai, 1834A, China) was water bathed for 30
min under fixed temperature (ranging from 10 to 50 oC, interval 5 oC) before each test,
which was repeated three times. The thermal denaturation curve was drawn with
temperature as horizontal ordinate and fractional viscosity as vertical coordinate,
when the fractional viscosity was 0.5, the corresponding point of horizontal ordinate
was just the Td.
The formula for the thermal denaturation curve was as below, ηsp represents the
specific viscosity, t represents the time STCC solution flow out the capillary, t0
represents the mixed solvent and ηspT represents the specific viscosity at each fixed
temperature (ranging from 10 to 50 oC, interval 5 oC).
ηsp = (t-t0) / t0
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Fractional viscosity = (ηspT-ηsp10oC) / (ηsp 50oC-ηsp10oC)

2.9.2. Differential Scanning Calorimetry (DSC)

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].

2.10. Scanning Electron Microscopy (SEM)

The morphological characteristics of STCC sponge were observed by SEM


(Hitachi, S4800, Japan). STCC sponge was prepared by freeze-dried in Vacuum
freeze dryer (Scientz Biotechnology Co., Ltd, Scientz-18ND, China). The surface and
cross-section of STCC samples were prepared by cut into approximately 1 cm×0.5 cm
rings, and then both were mounted on stubs, sputter-coated with gold by auto fine
coater (JEOL Ltd, JFC 1600, Japan) and then observed at 30, 100 and 300
magnifications [24, 25].

3. Results and Discussion

3.1. Optimization of Collagen Purification

3.1.1. Single Factor Test

Salting out is a method of separating proteins based on the principle that


proteins are less soluble at high salt concentrations. The salt concentration needed for
the protein to precipitate out of the solution differs from protein to protein. Various
parameters affect the optimization of the experimental conditions for the development
of salting out method for STCC purification. The salting out time, NaCl concentration
and STCC concentration are generally considered to be the most important factors
that affect the recovery yield (%) of STCC.

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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.

3.1.2. Orthogonal Test

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

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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.

3.2. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

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

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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.

3.3. UV Absorption Spectrum

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.

3.4. Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectrum of STCC exhibited the characteristics peaks of amide A, B as


well as I, II, and III was depicted in Fig. 4. The absorption peak of amide A was
observed at 3488 cm-1, which was caused by the NH band (hydrogen bond) stretching
vibration, and amide B had an absorption peak 2903 cm-1 because of CH asymmetric
stretching vibration, which occurs commonly in collagen. The amide I peak was
observed at 1622 cm-1 for the C=O stretching of polypeptide skeleton, the vibration
frequency of amide I was sensitive to changes of triple helix structure but not affected
by side groups of peptide chain. The absorption peak of amide II band was found at
1512 cm-1 because of the CN stretching vibration and NH bending vibration,
suggesting that there was stronger hydrogen bond in STCC. The amide III represented
the combination peaks between NH deformation and NH stretching vibration and was

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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].

3.5. Amino-Acid Composition

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

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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.

3.6. Effect of pH and NaCl on STCC Solubility

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].

3.7. Thermal behaviors


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Thermal stability influences on the durability of the collagen-based biomaterials.
Denaturation temperature is an important index for evaluating the thermal stability of
collagen [35]. As depicted in Fig. 6(A), the Td of STCC was calculated as about 35.1
o
C by the thermal denaturation curve when the fractional viscosity was 0.5. It was
higher than that of black carp (25.6 oC) [36], grass carp (24.6 oC) [37], brown backed
toadfish (28 oC) [38], yellowfin tuna (32 oC) [34], Amur sturgeon skin (32.46 oC) [23],
and sea cucumber (32.5 oC) [9], and similar to that of squid skin (34.80 oC) [28] and
horse-faced fish skin (34.99 oC) [33]. As previously reported by Nagai [13], Td of
different aquatic collagens would be affected by the animals habiting temperature and
the thermal stability would be influenced by the imino acid content which it related to
the collagen molecular structure [29]. The higher the imino acid content, the more
stability is obtained. In contrast, the Td of STCC was lower than that of calf skin
collagen (40.8 oC) and pig skin collagen (37 oC) [21]. However, the Td of collagen
from soft-shelled turtle skin was found to be 36 oC [13], which was almost the same
as that of porcine collagen, indicating that there were differences among collagens
from different tissues but the same animal. And this was consistent with the viewpoint
that the turtle collagens have higher Td than other fishery species and further
confirmed that STCC may have the superiority of higher thermal stability and is
promising as an advantage in biomedical applications [13].
The DSC pattern showed the endothermal peaks of STCC in Fig. 6(B), which
indicated that the Tm was 105.14 oC. In contrast, higher Tm of Amur sturgeon (115.42
o
C) was previously found by Wang et al. [23], which also demonstrated that thermal
behaviors of collagens correlates with environmental temperature. Generally, the
turtle collagen is less thermally stable than mammalian collagen. Despite its Td
superiority, it is still necessary to do structure modification such as cross-linking of
STCC to improve thermal behaviors [4].

3.8. Scanning Electron Microscopy (SEM)

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
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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. 2. SDS-PAGE of the calipash collagen from Pelodiscus sinensis. lane 1,


molecular weight marker; lane 2, crudely extracted collagen; lane 3, purified collagen.

Fig. 3. UV scanning spectrum of the calipash collagen from Pelodiscus sinensis.

Fig. 4. FTIR spectrum of the calipash collagen from Pelodiscus sinensis.

Fig. 5. Effects of pH and NaCl concentration on solubility of the calipash collagen


from Pelodiscus sinensis. A, pH; B, NaCl concentration

Fig. 6. Thermal behavior results of the calipash collagen from Pelodiscus sinensis. A,
thermal denaturation curve; B, melting temperature curve.

Fig. 7. Scanning electron microscopy of the calipash collagen from Pelodiscus


sinensis. A, surface; B, cross-section

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Fig.1

22
Fig.2

23
Fig.3

24
Fig.4

25
Fig.5

26
Fig.6

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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

Groups Salting out NaCl STCC Recovery yield of


Time Concentration Concentration STCC
(h) (M) (g/L) (%)
(A) (B) (C)
1 1 (12) 1 (2) 1 (6) 65.30
2 1 (12) 2 (3) 2 (8) 75.95
3 1(12) 3 (4) 3 (10) 73.39
4 2 (24) 1 (2) 2 (8) 80.47
5 2 (24) 2 (3) 3 (10) 78.47
6 2 (24) 3 (4) 1 (6) 60.73
7 3 (36) 1 (2) 3 (10) 73.50
8 3 (36) 2 (3) 1 (6) 77.54
9 3 (36) 3 (4) 2 (8) 71.54
K1 214.64 219.27 203.57
K2 219.67 231.96 227.96
K3 222.58 205.66 225.36
k1 71.55 73.09 67.86
k2 73.22 77.32 75.99
k3 71.19 68.55 75.12
R 2.03 8.77 8.13

30
Table 3 Amino acid composition of turtle calipash and collagen from calipash

Composition (%)
Amino acid
turtle calipash turtle calipash collagen

Asparatic acid 5.36 6.04


Threonine 2.55 2.88
Serine 4.55 4.35
Glutamic acid 11.51 11.51
Glycine 23.15 19.99
Alanine 12.76 11.38
Cysteine/2 0.10 0.00
Valine 2.13 2.50
Methionine 0.01 0.00
Isoleucine 1.42 1.69
Leucine 2.80 3.38
Tyrosine 2.45 0.00
Phenylalanine 0.97 0.47
Lysine 3.33 3.82
Histidine 0.82 0.94
Arginine 7.56 8.04
Tryptophan 0.02 0.01
Hydroxylysine 0.03 0.01
Proline 10.27 12.15
Hydroxyproline 8.22 10.84

31