LWT - Food Science and Technology 74 (2016) 145e153

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LWT - Food Science and Technology

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

Extraction and characterization of chicken feet soluble collagen

Cunshan Zhou a, Yanhua Li a, Xiaojie Yu a, Hua Yang b, Haile Ma a, *,
Abu ElGasim A. Yagoub c, Yu Cheng a, Jiali Hu a, Phyllis Naa Yarley Otu a, d
a

School of Food and Biological Engineering, Jiangsu Provincial Key Laboratory for Physical Processing of Agricultural Products, Jiangsu University, No. 301
Xuefu Road, Zhenjiang, 212013, China
b
College of Biological and Environmental Sciences, Zhejiang Provincial Top Key Discipline of Biological Engineering, Zhejiang Wanli University, No. 8 South
Qian Hu Road, Ningbo, 315100, China
c
Faculty of Agriculture, University of Zalingie, P.O. Box 6, Zalingie, Sudan
d
Science Laboratory Technology, Accra Polytechnic, P.O. Box 561, Accra, Ghana

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 24 January 2016
Received in revised form
5 July 2016
Accepted 8 July 2016
Available online 9 July 2016

Sodium chloride-soluble collagen (SSC), acetic acid-soluble collagen (ASC) and pepsin-soluble collagen
(PSC) were extracted from the skin of chicken feet and then characterized. PSC, ASC and SSC showed the
yields of 49.10%, 14.49% and 1.13% (Based on lyophilized dry weight), respectively. PSC, ASC and SSC were
characterized as type I collagen, containing a1 and a2 chains as well as b and g-chains. Circular dichroism
(CD) and Fourier transform infrared (FTIR) spectra of PSC, ASC and SSC were similar, suggesting that they
maintained their intact triple helical structure. PSC, ASC and SSC contained Gly as the major amino acid
with high contents of Glu, Ala, Pro and Hyp. Scanning electron microscopy (SEM) and atomic force
microscopy (AFM) images of PSC, ASC and SSC revealed that their surface topography were similar.
Dynamic elastic behavior in PSC, ASC and SSC was detected. PSC showed the largest elasticity. Temperature sweeps test indicated that PSC had the highest denaturation temperature, followed by ASC, and
then by SSC. Proline hydroxylation of PSC was higher (45.8%) than that of ASC, and SSC and accordingly
PSC showed the highest thermal stability. PSC showed the highest degree of hydrolysis compared to ASC
and SSC.
2016 Elsevier Ltd. All rights reserved.

Keywords:
Chicken feet skin
Collagen extraction
Characterization
Rheological properties
Degree of hydrolysis
Chemical compounds studied in this article:
Acetic acid (PubChem CID: 176)
Bromophenol blue (PubChem CID: 8272)
Hydrochloric acid (PubChem CID: 313)
Potassium dihydrogen phosphate (PubChem
CID: 516951)
Sodium hydroxide (PubChem CID: 14798)
SDS (PubChem CID: 3423265)
Sodium chloride (PubChem CID: 5234)
Tris hydrochloride (PubChem CID: 93573)
Urea (PubChem CID: 1176)

1. Introduction
Traditionally, collagen is extracted and characterized from byproducts of mammal and marine animals (Li, Mu, Cai, & Lin,
2009). However, China is currently one of the leading countries in
production of poultry. Chicken skin, a byproduct derived from
chicken meat processing, is highly underutilized, constituting huge
cost for waste disposal and danger to the environment (Feddern
et al., 2010). Several attempts have previously been made at

* Corresponding author.
E-mail address: mhl@ujs.edu.cn (H. Ma).
http://dx.doi.org/10.1016/j.lwt.2016.07.024
0023-6438/ 2016 Elsevier Ltd. All rights reserved.

developing novel chicken skin based products in order to diversify

its utilization and reduce waste (Onuh, Girgih, Aluko, & Aliani,
2014). However, an area of research that is yet to be explored is
the development of chicken skin based products with functional
and health promoting values. Chicken feet are mainly used to
produce animal feed and low-end meat products. Extraction and
characterization of collagen from chicken feet could effectively
increase its economic value-added and hence increase its
comprehensive utilization.
Type I collagen is the most abundant collagen in the organisms
and is mostly found in skin, tendon and ligament. It is characterized
by a unique right-handed triple helical structure with polypeptide
chains of a G-X-Y amino acids repeating sequence, with X and Y

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C. Zhou et al. / LWT - Food Science and Technology 74 (2016) 145e153

often represent hydroxyproline and proline (Nalinanon, Benjakul,

Hideki Kishimura, & Osako, 2011). Type I collagen has a wide
scope of applications, particularly in pharmaceutical, food and
biomedical industries (Yamauchi, & Sricholpech, 2012; Zhang,
Olsen, Grossi, & Otte, 2013).
Investigations on collagens extracted from the chicken feet are
scanty. There is growing interest in extraction of collagen from
chicken by-products by aid of acids (Cheng, Hsu, Chang, Lin, &
Sakata, 2009). However, no studies on hydrolysis and rheological
properties of collagens extracted by sodium chloride, acetic acid
and pepsin from the chicken feet skin are found in the literature
(Liu, Lin, & Chen, 2001). Accordingly, the objective of this paper was
to prepare and characterize sodium chloride-, acetic acid- and
pepsin-soluble collagens from the chicken feet skin. This paper can
provide basis for production of collagens, with potential commercial applications.
2. Materials and methods
2.1. Materials
Frozen chicken feet were purchased from a local market in
Zhenjiang, Jiangsu province, China. They were transported to the
Food Science laboratory, Jiangsu University within 30 min, and then
immediately frozen at
20  C and kept for further use. All chemicals used were of analytical grade.
2.2. Extraction of collagen
2.2.1. Pretreatment of chicken feet
The frozen chicken feet were rst thawed at 4  C and then a skin
was removed manually and cut into small pieces (1  0.5 cm). The
pieces were homogenized for 5 min at speed of 10,000 rpm using a
homogenizer (Nengu, NSR-I, China) placed in an ice-water bath.
The homogenate was soaked in a solution of 20% (w/v) NaCl in
0.05 M Tris-HCl (pH 7.5) at a ratio of 1:20 (w/v). The mixture was
centrifuged at 10,000g for 20 min by a centrifuge (Avanti J-26XP,
Beckman Coulter,USA) and the precipitate was washed repeatedly
with distilled water to remove fats and bubbles. The pretreated skin
sample was lyophilized and kept in a desiccator until use. The
extraction process is shown in Fig. 1 (Miller & Kent Rhodes, 1982).
2.2.2. Extraction of salt-soluble collagen (SSC)
The salt-soluble collagen (SSC) was prepared by soaking the
pretreated chicken feet skin in a salt solution (0.45 M NaCl in
0.05 M Tris-HCl, pH 7.5) at a ratio of 1:80 (w/v). Extraction mixture
was homogenized for 5 min at speed of 10,000 rpm, using the
homogenizer placed in the ice-water bath, and then left to stand for
48 h. At the end of extraction process, the mixture was centrifuged
at 17,000g for 30 min. The supernatant (SSC) and residue were kept
for further processing.
2.2.3. Extraction of acid-soluble collagen (ASC)
The acid soluble collagen from the skin of chicken feet was
isolated following the method of Nalinanon, Benjakul,
Visessanguan, and Kishimura (2008) with some modications.
Undissolved matter from salt soluble collagen extraction was
soaked in 0.5 M (w/v) acetic acid at a ratio of 1:80 (w/v). The
mixture was homogenized for 5 min as before and then left to stand
for 48 h. The mixture was centrifuged at 17,000g for 30 min. The
supernatant (ASC) and residue were kept for further processing.
2.2.4. Extraction of pepsin-soluble collagen (PSC)
The pepsin-soluble collagen was extracted according to
Nalinanon, Benjakul, Visessanguan, and Kishimura (2007). The

residual pellet from acid extraction was soaked in 0.5 M acetic acid
(pH 2) containing 0.1% (w/v) pepsin (extracted from porcine gastric
mucosa, EC 3.4.23.1; 4500 units/mg protein, Sigma-Aldrich Co.,
USA) at a ratio of 1:80 (w/v). The mixture was homogenized for
5 min as before and then left to stand for 48 h. The homogenate was
centrifuged at 17,000g for 30 min. The supernatant (PSC) was kept
for further processing.
2.2.5. Purication of collagen
Collagens extracted with the aid of sodium chloride, acetic acid,
and pepsin (supernatants of SSC, ASC and PSC) were puried by
salting-out overnight in 0.9 M NaCl (supernatant of SSC was acidied in 0.01 M HCl before salting-out). At the end of salting-out, the
supernatant was centrifuged at 2500g for 30 min. The precipitate
was dissolved in 1.0 M NaCl (prepared in 0.05 M Tris-HCl, pH 7.5).
The solution was centrifuged at 2500g for 30 min and the pellet was
removed. The resulting supernatant was salted-out overnight in
2.4 M NaCl and then centrifuged as before. The resulting pellet was
dissolved in 0.5 M acetic acid and subsequently dialyzed in 0.1 M
acetic acid for 24 h and in distilled water for 48 h. The puried SSC,
ASC and PSC were lyophilized and stored in a desiccator until use.
All operations were carried out at 4  C.
2.2.6. Determination of hydroxyproline content
The hydroxyproline content of lyophilized SSC, ASC and PSC
were determined according to the method of Bergman and Loxley
(1963) with a slight modication. A predetermined weight of
collagen sample was hydrolyzed with 6 M HCl at 110  C for 24 h in
an oil bath. The hydrolysate was claried with activated carbon and
ltered through Whatman No. 4 lter paper. The ltrate was
neutralized with 10 M and 1 M NaOH to obtain a pH of 6.0e6.5. The
neutralized sample (0.1 mL) was transferred into a test tube and
isopropanol (0.2 mL) was added and mixed well. An aliquot of
0.1 mL of oxidant solution [mixture of 7% (w/v) chlororamine T and
acetate/citrate buffer, pH 6, at a ratio of 1:4 (v/v)] was added and
mixed thoroughly. Then 1.3 mL of Ehrlichs reagent solution
[mixture of 2 g of p-dimethylamino-benzaldehyde in 3 mL 60% (v/v)
perchloric acid (w/v) and isopropanol at a ratio of 3:13 (v/v)] were
added. The mixture was agitated and heated at 60  C for 25 min in a
water bath and then cooled for 2e3 min in running water. The
solution was diluted to 5 mL with isopropanol. Absorbance was
read against water at 558 nm. A hydroxyproline standard solution,
with concentrations ranging from 10 to 60 ppm, was used. Hydroxyproline content was calculated and expressed as mg/g of
sample. The conversion factor for calculating the collagen content
from hydroxyproline of chicken
feet skin was 7.7
(Kittiphattanabawon, Benjakul, Visessanguan, Nagai, & Tanaka,
2005).
2.3. Characterization of collagen
2.3.1. Analysis of amino acids
The amino acids content was determined by hydrolyzing a
sample in 6 M HCl under vacuum at 110  C for 24 h. The hydrolysate
was dried by a vacuum concentrator at 60  C and then dissolved in a
citrate buffer (pH 2.2). The amino acid content was measured by an
automatic amino acid analyzer (Sykam S-433 D, Germany).
2.3.2. Sodium dodecyl sulphate polyacrylamide gel electrophoresis
(SDS-PAGE)
Protein patterns of SSC, ASC and PSC were determined according
to the method of LaemmLi (1970). Electrophoresis gels used were
12% separating gel and 5% stacking gel. The lyophilized collagen was
dissolved in 0.1 M Tris-HCl containing 1.0% (w/v) SDS and 3 M urea,
pH 6.8, at a ratio of 0.1% (w/v). The resulting solution was mixed

C. Zhou et al. / LWT - Food Science and Technology 74 (2016) 145e153

147

Fig. 1. Flow chart for the extraction procedures of SSC, ASC and PSC from the skin of chicken feet.

with the sample buffer at a ratio of 1:1 (v/v) and then denatured at
100  C for 3 min. Ten microliters of collagen solution was loaded
onto the gel. At the beginning, electrophoresis was run at 90 V until
the bromphenol blue tracking dye entered the separating gel.
Electrophoresis was continued at 100 V to the end of separation
process. After electrophoresis, the gel was stained with 0.05% (w/v)
Coomassie Brilliant Blue R-250 in 15% (v/v) methanol and 5% (v/v)
acetic acid and destained with 30% (v/v) methanol and 10% (v/v)
acetic acid. Separated collagen bands were compared with standard
molecular weight markers (Takara, SDS-3596Q).

12 h. Surface topography was performed by a nanoscope V electronics atomic force microscope (Bruker Inc. Germany). All images
were scanned in air using standard peak-force mode and a silicon
cantilever.

2.3.3. Fourier transform infrared (FTIR) spectroscopy

Infrared spectra of the lyophilized collagens were measured in
absorbance mode 4000e400 cm
1 on FTIR spectrophotometer
(Nicolet, Nexusn670). Samples were prepared by grinding collagen
with KBr and then pressed into a translucent pellet. Scanning was
performed with a resolution of 4 cm
1. The FTIR spectra were an
average of 64 scans and they were analyzed using OMNIC software
(Thermo Scientic, Madison, WI, USA).

2.3.6. Circular dichroism (CD) spectra

The collagen was dissolved in 0.01 M HCl to a concentration of
0.1 mg/mL and then the resulting solution was equilibrated in a
refrigerator (4  C) for 12 h (Lakra et al., 2014). The CD spectrum was
recorded in a wavelength range from 190 nm to 250 nm on JACSO J815 spectropolarimeter. Scanning was done (under nitrogen atmosphere, 20  C) at speed of 50 nm/min and a resolution of 0.5 nm.

2.3.4. Atomic force microscopy (AFM)

The lyophilized collagen was dissolved in 0.2 M acetic acid to a
concentration of 4 mg/mL. An aliquot of 5 mL sample was deposited
onto freshly cleaved mica and dried at room temperature (25  C) for

2.3.5. Scanning electron microscopy (SEM)

The morphological characteristics of the collagen samples were
observed with a scanning electron microscope (JSM-7001F, JEOL,
Tokyo, Japan). Before using the scanning electron microscope, the
sample was coated with a conductive layer of gold-palladium.

2.3.7. Rheological properties

The rheological properties for 1% (in 0.5 M acetic acid, w/v)
collagen solution were tested on a DHR-1 rheometer (TA Instruments, Surrey, UK) using a parallel-plate geometry (diameter
40 mm, gap 1000 mm). For precise control of sample temperature,

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C. Zhou et al. / LWT - Food Science and Technology 74 (2016) 145e153

an accuracy of 0.1  C was ensured in the Peltier temperature

controller. The cross-linking behavior of collagen was assessed by
dynamic frequency sweeps. The frequency sweeps for the collagen
solution was conducted within the linear viscoelastic region at a
constant strain of 5% and a frequency range between 0.1 and
10 Hz at 25  C (Lai, Li, & Li, 2008). The dynamic temperature sweeps
were conducted within the linear range at a constant strain of 5%
and a given frequency of 60 rad/s. The 1.0% (w/w) collagen solution
was heated from 10 to 70  C at a rate of 0.5  C/min. Changes in
storage modulus (G0 ), loss modulus (G00 ) and loss tangent
(tan d G00 /G0 ) were recorded.
2.3.8. Simulated gastrointestinal digestion
Digestion of the collagen samples was performed according to
the U.S Pharmacopeial method (United States Pharmacopeial
Convention Council of Experts, 2007). Simulated gastric uid was
prepared by dissolving pepsin in 0.32% salt solution (w/v). The pH
of gastric uid was adjusted to 1.2 0.1 with HCl, and then heated
for 10 min at 37  C (Chen, Muramoto, & Yamauchi, 1995). Simulated
intestinal uid was prepared by dissolving trypsin in 0.05 M
KH2PO4 (1:100, w/v). The pH was adjusted to 6.8 with NaOH and
then heated as before. The collagen was rst digested in the
simulated gastric uid (at a ratio of 1:50, w/v) for 4 h at 37  C. Then,
the digested gastric mixture was added immediately to the simulated intestinal uid (at a ratio of 1:100, w/v), and left to stand for
6 h at 37  C. The digestion process was terminated by heating the
mixture in boiling water for 10 min. The mixture was cooled to
room temperature (25  C) and centrifuged at 10,000g for 20 min.
The degree of hydrolysis (DH) of collagen was measured according
to the method of Adler-Nissen (1979) with slight modication
(Zhou et al., 2015).
2.4. Statistical analysis
Experiments were done in triplicate and data obtained were
subjected to one-way ANOVA using Origin Pro 8.0. Differences
among means were considered signicant at p < 0.05.
3. Results and discussion
3.1. Collagen properties
In Fig. 2a, yields of SSC, ASC and PSC from the chicken feet skin
were 1.13, 14.49 and 49.10%, respectively. The yield of ASC from
chicken feet skin is comparable to that from bigeye snapper skin
(Kittiphattanabawon et al., 2005). Results indicate that the yield of
collagen is noticeably increased (P < 0.05), approximately by 3.4fold when pepsin is used to extract the residual collagen after acid
extraction. Therefore, pepsin is effective in extracting collagen from
the skin of chicken feet. This result agrees with those found in
pepsin-soluble collagens extracted from the skins of chicken (Lin,
Loughran, Tsai, & Tsai, 2013), Pharaoh Cuttlesh (Nalinanon,
Benjakul, Kishimura, & Osako, 2012), threadn bream (Nalinanon
et al., 2008), and bigeye snapper (Nalinanon et al., 2007). Pepsin
has been reported to solubilize collagen in the telopeptide region,
by cleaving some peptide bonds resulting in a higher efcacy of
collagen extraction (Jridi et al., 2015). In this respect, the PSC
extraction is possibly more suitable for industrial production.
The subunit compositions of collagens from the chicken feet
skin are visualized in the electrophoretogram (Fig. 2b). SSC, ASC
and PSC are mainly composed of at least two different a-chains (a1
and a2) and b-chains (dimmers of the a-chains) and g-chains (trimers of the a-chains). These results suggest that type I collagen is
the main component of the chicken feet skin, coinciding with the
ndings reported in the literature (Chen, Ma, Zhou, Liu, & Zhang,

2014; Mori et al., 2013). Collagen fragments with molecular

weights lower than a-chains are not observed in the electrophoretogram of SSC, ASC and PSC. This indicates that all extracted
collagens maintained their structural integrity (Jongjareonrak,
Benjakul, Visessanguan, Nagai, & Tanaka, 2005).
The major peaks of FTIR spectra of SSC, ASC and PSC are presented in Fig. 2c and the corresponding denotation of the peaks
(Liang et al., 2014a) is shown in Table 1. The collagens from chicken
feet skin exhibit FTIR spectra similar to that found in other skin
collagens (Duan, Zhang, Du, Yao, & Konno, 2009; Liu, Li, & Guo,
2007; Matmaroh, Benjakul, Prodpran, Encarnacion, & Kishimura,
2011). In which the absorption characteristic peaks in the spectra
are situated in the amide band region including amide A and B as
well as amide I, II and III. The collagens from chicken feet skin are
similar in characteristic absorption frequencies but they show
discrepancy in the intensity.
Absorption characteristic of amide A, commonly associated with
NeH stretching vibration, locates in the wavenumber range
3400 cm
1 to 3440 cm
1 (Sai & Babu, 2001). In general, all collagens are mainly stabilized by hydrogen bond as shown by a lower
frequency (3286e3308 cm
1), compared with a free NeH
stretching vibration (3400e3440 cm
1) (Kittiphattanabawon,
Benjakul, Visessanguan, & Shahidi, 2010). The amide A peak of
PSC (3308 cm
1) is higher than that of SSC (3286 cm
1) and ASC
(3297 cm
1). This higher absorption band suggests that less NeH
groups in PSC are involved in hydrogen bonding in polypeptide
chain. When NeH group is involved with hydrogen bond in peptide
bonds, the position starts to shift to lower frequency (Matmaroh
et al., 2011). Amide B band of the collagens (2924e2932 cm
1) is
related with asymmetrical stretch of CH2 stretching vibration and
absorption due to the eCH2 alkyl chain (Hsu, Weng, Liao, & Chen,
2005).
The amide I, with characteristic wavenumber in the range of
1600e1700 cm
1, is located in the double bond region and usually
not affected by the side group peptide. Amide II is generally
responsible for the combination of the NeH in-plane bend and the
CeN stretching vibration (Barth & Zscherp, 2002). The vibration
frequencies of amide I and amide II bands have positive correlation
with the degree of molecular order (Muyonga, Cole, & Duodu,
2004). The amide I and II absorptions are observed at 1629 and
1548 cm
1 for PSC, at 1630 and 1552 cm
1 for ASC and at 1630 and
1550 cm
1 for SSC, respectively. It can be seen that amide I and II
bands for PSC are slightly lower than those for ASC and SSC, suggesting a lower degree of molecular order for PSC. Consequently,
the triple-helical structure might be slightly affected by pepsin
digestion during collagen extraction, whereas the secondary
structure of resulting PSC might be altered to some degree. Similar
changes in amide I and II bands has been reported on acid soluble
and pepsin soluble collagens extracted from cartilages of brownbanded bamboo shark and blacktip shark (Kittiphattanabawon
et al., 2010). The results of amide I and II absorptions are in accordance with SDS-PAGE electrophoretogram of the collagens
extracted from chicken feet skin (Fig. 2b). Amide III bands are found
at wavenumber of 1237, 1238 and 1242 cm
1 for SSC, ASC and PSC,
respectively. The amide III peak consists components from CeN
stretching and NeH in plane bending from amide linkages, as well
as absorptions arising from wagging vibrations from CH2 groups
from the glycine backbone and proline side-chains (Plepis, Goissis,
& Das Gupta, 1996), indicating that hydrogen bonds are involved in
SSC, ASC and PSC.
SSC, ASC and PSC show absorptions at 1082, 1079 and
1081 cm
1, respectively, arising from the CeO stretching vibrations
of the carbohydrate moieties attached to the protein (Petibois,

le

ris, 2006). This result suggests
Gouspillou, Wehbe, Delage, & De
that the collagens might contain carbohydrates, which are attached

C. Zhou et al. / LWT - Food Science and Technology 74 (2016) 145e153

149

Fig. 2. Yield (a), electrophoretogram (M, Molecular weight markers) (b), FT-IR spectra (c) and CD spectra (d) of SSC, ASC and PSC from the skin of chicken feet.

Table 1
Fourier transform infrared spectra peak locations and assignment for collagens from the skin of chicken.
Region

Peak wavenumber (cm
1)

Assignment

SSC

ASC

PSC

3286
2924
3082

3297
2930
3080

3308
2932
3083

NeH stretching vibration coupled with H-bond

asymmetrical stretch of CH2
asymmetrical stretch of CH2

1630
1550
1456

1630
1552
1454

1629
1548
1452

C]O stretching vibration/H-bond coupled with COO

NeH bend coupled with CeN stretch
CH2 bend

1398

1402

1405

COO
symmetrical stretch

Amide A
Amide B
e
Amide
Amide

e
Amide
e

1337

1339

1337

CH2 wag

1237
1082

1238
1079

1242
1081

NeH bend coupled with CeN stretch

CeO stretching vibration

to hydroxylysine residues of the polypeptide chain by O-glycosidic

bonds. The absorption peaks in the range 1460 cm
1 to 1240 cm
1
depicts that the triple helical structure of collagen is intact (Nagai &
Suzuki, 2000). As a conclusion, the FTIR peak locations (Table 1)
indicate that the inherent characteristics of SSC, ASC and PSC are
conserved.
Moreover, CD spectra of SSC, ASC and PSC show a positive peak
at 213 nm and a negative peak at 198 nm with a consistent crossover point at 213 nm (Fig. 2d), conrming presence of specic
feature of the triple helical structure of collagen (Liang et al.,
2014b). Except for the change in amplitude, the red shift of negative peak and the vanishing of positive peak are not observed,
ensuring existence of the triple helical structure in all collagens.
3.2. Amino acids composition
Amino acids proles of SSC, ASC and PSC have great similarity in
terms of residues content (Table 2). Except for SSC, Gly represents
the most abundant amino acid in all collagens, accounting for more

than one third of the total amino acid residues. The Gly content in
PSC is higher than in ASC and SSC. The lowest content of Gly in SSC
is possibly ascribed to presence of impurities with protein molecules that have not been entirely removed during salt extraction
process. Gly contributes in formation of the nal collagen superhelix, allowing the three helical a-chains to pack tightly together
(Alberts et al., 2002). The collagen samples also contain high
amounts of Glu, Ala, Pro and Hyp. The imino acid contents (Pro and
Hyp) of ASC and PSC from the chicken feet skin (210 and 216 residues/1000 residues, respectively) are relatively higher than those
from skin of cuttlesh (Nalinanon, Benjakul, Kishimura, & Osako,
2012), carp (Duan et al., 2009), and bigeye snapper
(Kittiphattanabawon et al., 2005). The imino acids content plays an
important role in the integrity of collagen construction. Pro contributes to the composition of peptide primary structure and stabilization of collagen tertiary structure. Hyp plays role in formation
of hydrogen bond through its eOH group. Consequently, the total
imino acids content, especially Hyp, and the degree of proline hydroxylation are important in the context of thermal stability and

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C. Zhou et al. / LWT - Food Science and Technology 74 (2016) 145e153

Table 2
Amino acids prole of the collagens extracted from chicken feet skin (residues/
1000 residues).

ASP
Thr
Ser
Glu
Gly
Ala
Val
Met
Ile
Leu
Phe
His
Lys
Arg
Pro
Hyp
Imino acidsa
Proline hydroxylation (%)b
Total
a
b

Pro plus Hyp.

Hyp/(Pro plus Hyp).

SSC

ASC

PSC

42
37
40
99
239
107
39
10
34
60
25
15
58
51
90
54
144
37.5
1000

29
22
24
80
336
128
20
2
12
27
15
6
38
51
115
95
210
45.2
1000

28
19
23
79
344
127
19
5
11
24
12
5
38
50
117
99
216
45.8
1000

functionality of collagen-derived gelatin (Bae et al., 2008; GomezGuillen et al., 2002). The degree of proline hydroxylation of PSC,
ASC and SSC from the chicken feet skin is 45.8, 45.2 and 37.5%,
respectively. A higher hydroxylation of prolyl residue is correlated
to a higher denaturation temperature (Matmaroh et al., 2011).
Accordingly, PSC is characterized by high thermal stability and good
gelling attributes, followed by ASC and then by SSC. These ndings
agree with the results of temperature sweep test depicted in Fig. 5a
and b.
3.3. Structure analysis
3.3.1. SEM analysis
In order to observe differences in the microstructures, SEM
analysis was employed. The SEM images (magnication of 300) of
SSC, ASC and PSC are depicted in Fig. 3aec. The surface morphology
of SSC, ASC and PSC is similar, looking like white cotton with loose
ber structure exhibiting corrugated sheet linked by irregulartwined ber ends. The coarse surface might be related to the
dehydration which leads to the condensation of polymer (Edidin,
2001; Zhang et al., 2015). Moreover, PSC has the least bril-like
lament owing to breakdown of non-helical ends by pepsin
upler, Gibis, Koholus, & Weiss, 2015).
enzyme (Oechsle, Ha

Fig. 3. SEM and AFM images of collagens extracted from the skin of chicken feet. SEM image: SSC: a, ASC: b and PSC: c. AFM morphology photos (SSC: d, ASC: e and PSC: f) and their
corresponding three-dimensional photos (SSC: g, ASC: h and PSC: i).

C. Zhou et al. / LWT - Food Science and Technology 74 (2016) 145e153

Fig. 4. The storage modulus G0 (a), loss modulus G00 (b) and loss tangent tan d (c) in
dynamic frequency sweep test of SSC, ASC, and PSC.

3.3.2. AFM analysis

The atomic force microscope images of collagens are depicted in
Fig. 3dei. Surface morphology characteristics and nanostructures
observed by AFM analysis agree with the SEM test. As can be seen,
SSC, ASC and PSC presented obvious and bulky ber bundle
structure (Fig. 3d, e and f). None of the collagen monomers can be

151

Fig. 5. The storage modulus G0 (a), loss modulus G00 (b) and loss tangent tand (c) in
dynamic temperature sweep test of SSC, ASC, and PSC.

observed within the visual range recorded, indicating that the

collagen molecules assembled into ber bundle (Cleaver & Looi,
2007; Ikoma, Kobayashi, Tanaka, Walsh, & Mann, 2003). The
evenly distributed porous network structure may be caused by
lamentous collagen bers. These bers gathered further and

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C. Zhou et al. / LWT - Food Science and Technology 74 (2016) 145e153

stabilized by covalent bonds.

3.4. Dynamic oscillatory analysis
3.4.1. Frequency sweeps
Dynamic frequency sweep tests are used to determine the frequency dependence of the storage modulus (G0 ), loss modulus (G00 )
and loss tangent (tan d). Fig. 4aec shows the dynamic frequency
sweep tests for the collagens extracted from chicken feet skin. It is
found that the deformation of polymers with external force is
tightly related to the storage modulus (Oechsle, Wittmann, Gibis,
Kohlus, & Weiss, 2014). However, G0 and G00 values of all collagens
increase with the increase of frequency from 0.2 to 10 Hz (Fig. 4a
and b). It is noticeable that the storage modulus is higher than the
loss modulus in all collagens, suggesting a greater contribution
from the elasticity than the viscosity. The loss modulus reects the
dissipated energy as a characteristic of the viscous properties, and
hence mechanical loss of polymers (Friess & Schlapp, 2001). PSC
displayed the largest dynamic elasticity and form junction zones
faster, resulting in strong consolidation of the network structure.
On the other hand, the tan d values crossed the threshold from
solid-like to liquid-like behavior (tan d 1) (Zhang, Chen, Li, & Du,
2010). The smaller the value of tan d, the more elastomeric or
rubbery is the behavior of the material (Korhonen, Hellen,
Hirvonen, & Yliruusi., 2001). Fig. 4c shows the changes of tan d as
a function of frequency. The tan d values of the collagens decrease
rapidly with the increase of frequency from 0.1 to 2 Hz, and
thereafter the tan d values decrease slowly. At frequency >2 Hz, all
collagens have tan d values less than one, proving higher elastic
modulus G0 than the viscous modulus G00 . In conclusion, the elastic
behavior characterizes the three collagen samples.
3.4.2. Temperature sweeps
As can be seen in Fig. 5a and b, the values of storage modulus G0
and viscous modulus G00 of all collagens decrease slightly with the
increase in temperature and fell suddenly at 37e44  C. Then the
values are kept relatively constant, reecting the typical denaturation temperature range. The G00 values of the collagens show a
sharp transition at higher temperature, which means higher thermostability (Safandowska & Pietrucha, 2013; Santos, Calero,
 oz, 2015). Results of the temperature sweep test
Guerrero, & Mun
indicate that the denaturation temperature of PSC is the higher,
followed by ASC and then by SSC. Fig. 5c shows the changes of tan d
as a function of temperature. The tan d values are less than one for
all collagens, which indicates a predominant elastic behavior.
3.5. Degree of hydrolysis (DH)
At the rst hour of gastric digestion phase, the DH of SSC, ASC
and PSC, increase rapidly to values ranging between 10.81 and
11.72% and thereafter it increase, with a decreasing rate, to the end
of the phase (i.e. 4 h). During the 6 h-intestinal digestion phase, the
DH of the collagens increase rapidly at the rst hour and then the
digestion increase slowly with prolonging the time. At the end of
the digestion process, the DH of PSC is higher (24.00%) than that of
ASC and SSC (Fig. 6). This is probably attributed to presence of more
exposed cleavage sites in the PSC molecules, resulting from the
action of pepsin enzyme during extraction process.
4. Conclusion
Salt-soluble (SSC), acid-soluble (ASC) and pepsin-soluble (PSC)
collagens extracted from the skin of chicken feet were classied as
type I. Efciency in collagen extraction from the skin was enhanced
by 3.4 folds over ASC when pepsin was used. PSC exhibited similar

Fig. 6. The dynamic changes of hydrolysis degree of SSC, ASC and PSC during simulated gastrointestinal digestion.

molecular properties to ASC and SSC with predominant triple helical structure, with high content of imino acids. All collagens were
similar in surface topography (SEM and AFM images) and FTIR
spectra. Rheological properties and simulated gastrointestinal
digestion of the three collagens were different. The higher thermal
stability of PSC indicated its potentiality as a substitute for
mammalian collagen. The skin of chicken feet could be a promising
source for collagen particularly that extracted with the aid of
pepsin.
Acknowledgements
The work was funded by the National High-tech Research and
Development Program (2013AA102203-02), Prospective Research
and Natural Science Foundation of Jiangsu Province
(BY2016020410, BK20130494), China Postdoctoral Science Foundation (2014T70489, 2013M541621, 1302152C), the Zhejiang Provincial Top Key Discipline of Bioengineering (KF2014006), Six talent
peaks project in Jiangsu Province (2015- NY-016), Foundation for
the Eminent Talents and Research Foundation for Advanced Talents
of Jiangsu University (12JDG074) and the Project Funded by the
Priority Academic Program Development of Jiangsu Higher Education Institutions (2013).
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