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Received: 25 August 2019    Revised: 24 January 2020    Accepted: 28 January 2020

DOI: 10.1111/jfbc.13256

FULL ARTICLE

Biochemical and structural characterization of sturgeon fish

skin collagen (Huso huso)

Maryam Atef1,2  | Seyed Mahdi Ojagh1  | Ali Mohammad Latifi3  |

Mina Esmaeili4  | Chibuike C. Udenigwe2

1
Department of Seafood Science and
Technology, Faculty of Fisheries and Abstract
Environmental Science, Gorgan University of The potential use of sturgeon fish skin waste (Huso huso), an Iranian major sturgeon
Agricultural Sciences and Natural Resources,
Gorgan, Iran species, as a rich source for collagen extraction was evaluated. Yields of ASC and PSC
2
School of Nutrition Sciences, Faculty of obtained by acidic and enzymatic extractions were 9.98% and 9.08% (based on wet
Health Sciences, University of Ottawa,
weight), respectively. SDS-PAGE profiles of both collagens led to classification of the
Ottawa, ON, Canada
3
Applied Biotechnology Research Center,
proteins as type I with two different α chains (α1 and α2). Scanning electron micros-
Baqiyatallah University of Medical Sciences, copy (SEM) of the collagen sponges indicated dense sheet-like film linked by random-
Tehran, Iran
4
coiled filaments. Glycine was the most predominant amino acid, and the imino acids
Department of Fisheries, Faculty of Animal
Sciences and Fisheries, Sari Agricultural contents were 21.14% and 21.58% for ASC and PSC, respectively. Fourier-transform
Sciences and Natural Resources University, infrared spectra (FTIR) confirmed that pepsin digestion did not disrupt PSC triple heli-
Sari, Iran
cal structure. Denaturation and melting temperatures of ASC and PSC were 29.34°C,
Correspondence 92.03°C, and 29.89°C, 88.93°C, respectively. Thus, the sturgeon fish skin waste
Seyed Mahdi Ojagh, Department of Seafood
Science and Technology, Faculty of Fisheries could serve as an alternative collagenous source for biomedical materials, food, and
and Environmental Science, Gorgan pharmaceutical applications.
University of Agricultural Sciences and
Natural Resources, Gorgan, Iran. Practical applications
Email: ojagh@gau.ac.ir, mahdi_ojagh@yahoo.
com
Beluga (Huso huso) is one of the most important sturgeon fish on the Caspian Sea and
aquaculture industries. With the exception of the meat and caviar, wastes generated
Chibuike C. Udenigwe, School of Nutrition
Sciences, Faculty of Health Sciences, after their processing are usually discarded. Skin and cartilage of sturgeon fish are
University of Ottawa, Ottawa, Ontario, K1H
the by-products of the processing, and they are often discarded as waste or used
8M5, Canada.
Email: cudenigw@uottawa.ca for low-value purposes, although they are a good source for production of collagen-
based biomaterials. Collagen type I is the most abundant collagen in the skin and this
work reports the sturgeon fish skin as an important collagen resource with potential
for use in the food, biomedical, and cosmetic industries.

KEYWORDS

amino acid, collagen, protein structure, skin waste, sturgeon fish

1 |  I NTRO D U C TI O N physical and functional properties. Collagen is widely utilized in

Collagen is the major component of the extracellular matrix and
the most predominant fibrous protein in the vertebrates. Collagen
has extensive industrial applications in food, leather, cosmetic,
biomedical and pharmaceutical products, due to its unique
the pharmaceutical and biomedical industries for purposes such
as drug delivery, management of hypertension, tissue engineer-
ing scaffold, blood valve prosthesis, dermal filler, wound dress-
ing, and inhibition of angiogenic diseases and arthritis (Atef &
Ojagh, 2017). Bovine and pig skins or chicken wastes are the most

J Food Biochem. 2020;00:e13256. wileyonlinelibrary.com/journal/jfbc |

© 2020 Wiley Periodicals LLC.     1 of 10
https://doi.org/10.1111/jfbc.13256
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2 of 10       ATEF et al.
available commercial collagens. However, the prevalence of some
diseases such as transmissible spongiform encephalopathy, bo-
vine spongiform encephalopathy, and the foot-and-mouth disease
(Jongjareonrak, Benjakul, Visessanguan, Nagai, & Tanaka,  2005),
and also prohibition by some religions (Tamilmozhi, Veeruraj, &
Arumugam, 2013), have limited the use of collagen from these
sources. Marine collagens, especially from seafood processing
by-products, are now explored as replacements for mammalian
collagen for use in different biomedical applications (Subhan,
Ikram, Shehzad, & Ghafoor, 2015).
Most of the protein-rich by-products of fish processing, such as
skin, scale, and bone, are discarded without any attempt at recov-
ery, which can cause environmental issues. Collagen derived from
fish processing by-products, especially skin and bone, can be widely
used as scaffolds and carriers, due to their desirable attributes in-
cluding low antigenicity, high biodegradability, biocompatibility, and
cell growth inducing potential. Also, many processors are no longer
allowed to discard offal waste directly into the sea, and this results in
additional expenses for refining the materials before disposal. Thus,
utilization of the by-products and their value addition are encour-
aged to protect the environment.
The great sturgeon or beluga (Huso huso) is one of the most im-
portant sturgeon fish on the south coast of the Caspian Sea and in
aquaculture industries. They are caught primarily for the meat and
caviar, which have high monetary value in the world market; how-
ever, wastes generated after their processing are usually discarded,
except for the swim bladder and notochord that are used in soups
and nutritional supplements (Ovissipour et al., 2009). Because of
the long time it takes for caviar production, the cost of culturing
sturgeon fish is higher than the other fish species. Furthermore, the
lack of utilization of sturgeon by-products limits the development
of its culture. The skin, cartilage, and fin of sturgeon fish are good
sources for collagen-based biomaterials production, which will lead
to increase in the economic viability of sturgeon aquaculture, and
reduction of environmental pollution associated with the waste dis-
posal (Zhang et al., 2014). Therefore, the goals of this study were to
isolate collagen from sturgeon fish skin by-products and study their
structural and biochemical properties related to food and biomate-
rial applications.
2 |  M ATE R I A L S A N D M E TH O DS
2.1 | Materials
Tris (hydroxymethyl) amino-methane, acetic acid, and pep-
sin (EC3.4.23.1; 10 FIP-unit/mg) from porcine gastric mucosa,
Coomassie Brilliant Blue R-250, sodium dodecyl sulfate (SDS), and
β-mercaptoethanol (βME) were purchased from Merck (Darmstadt,
Germany). High-molecular-weight marker was obtained from Bio-
Rad Laboratories (Hercules, CA, USA) and N,N,N,N-tetramethyl
ethylene diamine (TEMED) was purchased from Sigma-Aldrich (St.
Louis, MO, USA).
2.2 | Extraction of collagen
2.2.1 | Pretreatment of the fish skin
In order to eliminate non-collagenous proteins, small pieces of stur-
geon fish skin (Huso huso) (1.0 × 1.0 cm2) were immersed in 0.5 M of
NaOH at a ratio of 1:10 (w/v) with a continuous stirring for 48 hr. The
alkali solution was replaced after 24 hr, and then, were rinsed with
distilled water. Thereafter, the skin was defatted with 10% (w/v) of
butyl-alcohol at a ratio of 1:10 for 48 hr, and was later thoroughly
rinsed with distilled water to natural pH.
2.2.2 | Isolation of ASC and PSC
ASC and PSC were extracted as per reported method by Kittiphattana­
bawon, Benjakul, Visessanguan, Kishimura, and Shahidi (2010), with
a slight modification. Pretreated skin samples were combined with
0.5 M (w/v) acetic acid at a ratio of 1:15 for 48 hr. To eliminate undis-
solved debris, the sample solution was filtered with two layers of
cheesecloth, and then, the combined extracted was salted out by
the addition of NaCl to the final concentration of 2.6 M, in the pres-
ence of 0.05 M of Tris-HCl (pH 7.5). Thereafter, the precipitate was
collected by centrifugation at 10,000g for 30 min at 4°C, using a re-
frigerated centrifuge (Hermle, Z 36 HK, Germany). The solution ob-
tained was dialyzed (dialysis membrane with molecular weight cutoff
of 30 kDa) with 0.1 M of acetic acid for 24 hr, and then, further dia-
lyzed against distilled water for 48 hr. Final solution was lyophilized
to obtain the ASC powder.
After acid extraction, undissolved residue was utilized to extract
pepsin solubilized collagen in the presence of 0.5 M acetic acid and
20 unit/g pepsin for 48 hr. The preparation procedures of PSC were
performed by the same method as the ASC. The extraction yield
of both collagen samples was calculated according to the equation:
Yield (% ) = M∕M0 × 100
M and M 0 are the weight of lyophilized collagen (g) and the weight
of wet fish skin used (g), respectively.
Sturgeon fish skin contains the collagen at the level of 58 g/100 g.
Therefore, the collagen production yield based on the initial collagen
presented in the skin or recovery (%) was calculated as:
Production yield (based on the initial collagen in the fish skin) =
Production yield (based on the wet weight of fish skin) (%)/58%
2.3 | Sample characterization
2.3.1 | SDS-polyacrylamide gel electrophoresis
Protein patterns of collagen samples were profiled by the method re-
ported by Laemmli (1970) with 10% of resolving gel and 4% of stack-
ing gel. In order to prepare ASC and PSC (1 mg/ml), 0.02 M of sodium
ATEF et al.
phosphate buffer (pH 7.2) with 3.5 M of urea and 1% (w/v) SDS were
added to the samples followed by gently stirring at 4°C for 24 hr. Then,
the mixtures were centrifuged at 5,000g for 5 min. The supernatants
were blended with loading buffer containing 0.5 M of Tris-HCl (pH
6.8), 20% (v/v) glycerol, 4% (w/v) SDS, 10% (v/v) β-ME, and the mixture
heated to boiling for 5 min. Electrophoresis was conducted at 20 mA
and, thereafter, the gel was fixed with 0.05% (w/v) Coomassie brilliant
blue R-250 solution dissolved in 10% of acetic acid, 50% of methanol,
and 0.1% of water. The molecular weights of samples were determined
using a high molecular weight protein marker.
2.3.2 | Analysis of amino acid compositions
The amino acid composition of collagen samples was determined at
the Hospital for Sick Children, Proteomics, Analytics, Robotics and
Chemical Biology Center (SPARC BioCenter, Toronto, ON, Canada).
Lyophilized collagens were dissolved with 6 M of HCl solution for
24  hr at 110°C and analysis was conducted following a previously
reported method (Mohan & Udenigwe, 2015).
2.3.3 | Scanning electron microscopy (SEM)
The microstructures of collagens were observed by scanning electron
microscope (S4800, Hitachi, Tokyo, Japan). The isolated collagens were
mounted on a standard SEM sample holder, covered with gold ion, and
then, observed for surface morphology at different magnifications.
2.3.4 | Fourier transform-infrared spectroscopy
(FTIR)
The infrared absorption spectra of the lyophilized samples were acquired
using a Bruker Equinox 55 FTIR spectrometer (Ettlingen, Germany) with
an absorption mode at 4 cm−1 intervals in the 500–4,000 cm−1 range.
2.3.5 | Differential scanning calorimetry (DSC)
Thermal analysis of ASC and PSC was assessed by DSC (METTLER
TOLEDO, Switzerland). Lyophilized samples were rehydrated using
0.05 M of acetic acid solution at a ratio of 1:40 (w/v) for 2 days, and
then, sealed in aluminum pans (Jongjareonrak, Benjakul, Visessanguan,
& Tanaka, 2005) while the empty pan was applied as a reference. The
pans were scanned at the temperature range of 20°C–150°C at a heat-
ing rate of 1°C/min and with liquid nitrogen as the cooling medium.
2.3.6 | Ultraviolet spectra analysis
UV absorption spectrum of the collagen samples were recorded
using a microplate spectrophotometer (Tecan M1000, Männedorf,
|
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Switzerland). The collagen samples (1 mg/ml) were dissolved in
0.5 M of acetic acid solution. UV spectrum was recorded by scan-
ning the wavelength from 200 to 500 nm at the rate of 2 nm/min.
2.3.7 | Zeta potential analysis (ζ)
ζ-potential of ASC and PSC (1 ml) was evaluated using a Zetasizer
Nano Series Nano-ZS (Malvern Instruments Ltd., Malvern, UK). The
fish collagen samples (0.05 mg/ml) were mixed in 0.5 M of acetic acid
solution at 4°C for 24 hr. The samples were adjusted to different pH
values (2–11) and the isoelectric point (pI) was estimated from pH
zero.
2.3.8 | Solubility studies
Solubility of the collagen samples was evaluated at pH 1–10 and
different NaCl concentrations (0%–12%) using the procedure of
Montero, Jimennez-Colmenero, and Borderias (1991), with some
modifications in the sample preparation.
Effect of pH
Lyophilized collagens were suspended in 0.5 M of acetic acid solu-
tion (3 mg/ml; 8 ml) and the volume of the mixtures was made up to
10 ml with distilled water before adjusting the pH (1–10). The sam-
ples were stirred continuously for 30 min and centrifuged at 10,000g
for 30 min at 4°C. In order to measure the protein content of the su-
pernatant, bovine serum albumin (BSA) was used as a protein stand-
ard (Lowry, Rosebrough, Farr, & Randall, 1951).
Effect of NaCl
Lyophilized collagen samples were dissolved in 0.5 M of acetic
acid (6 mg/ml; 5 ml) at different concentrations (0%–12%, w/v) and
continuously stirred for 30 min. The samples were centrifuged at
10,000g for 30 min at 4°C and the protein content of the super-
natant was determined as previously reported (Lowry et al., 1951).
2.4 | Statistical analysis
All the tests were done in triplicate and the results were expressed
as mean ± SD.
3 | R E S U LT S A N D D I S CU S S I O N
3.1 | Extraction yield of ASC and PSC from the fish
skin
Yield of collagen samples from sturgeon fish skin by-products was
9.98% and 9.08% (wet weight basis), respectively. Collagen was
not thoroughly extracted with acetic acid solution, while limited
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pepsin enzyme digestion cleaved the intermolecular cross-links

without damaging in the triple helix structure at the telopeptide
area of collagen (Singh, Benjakul, Maqsood, & Kishimura, 2011).
The results obtained showed that the covalent bonds of collagen
molecules, by condensation of aldehyde groups at the telopeptide
area, could lead to the reduction of collagen solubility in the ASC
(Foegeding, Lanier, & Hultin, 1996), while the collagen solubility
increased after adding pepsin to acid solution (Jongjareonrak,
Benjakul, Visessanguan, Nagai, & Tanaka, 2005). Final yield of
both collagens was 19.06% (wet weight basis), which is higher
than values reported for collagens derived from bamboo shark
(18.24%), cobia (16.7%), catfish (12.8%), brownstripe red snapper
(13.7%), eel fish (13.7%), and bigeye snapper (7.5%) (Jongjareonrak,
Benjakul, Visessanguan, & Tanaka, 2005; Jongjareonrak, Benjakul,
Visessanguan, Nagai, & Tanaka, 2005; Kittiphattanabawon,
Benjakul, Visessanguan, Kishimura, & Shahidi  2010; Singh
et  al.,  2011; Veeruraj, Arumugam, & Balasubramanian,  2013;
Zeng et al., 2012). The yield differences can be due to the vari-
ation in sample preparation methods and also unique collagen
structure in the different species. The yield based on the initial
collagen presented in the skin or recovery (%) for both collagens
(ASC and PSC) was 32.86%, which is significantly higher than
those of dry tilapia fish skin (19.80% for acetic acid method and
20.03% for pepsin method) (Li et al., 2018), unicorn leatherjacket
skin (18.47%) (Ahmad, Benjakul, & Nalinanon,  2010), deep-sea
redfish skin (10.3%) (Wang et al., 2008), and bigeye snapper skin
(1.59%) (Kittiphattanabawon, Benjakul, Visessanguan, Nagai, &
Tanaka, 2005). However, the yield was lower than values reported
for tilapia skin collagen (53.4%) (Bi et al., 2019) and grass carp skin
(46.6%) (Wang, Liang, Wang, et al., 2014). F I G U R E 1   SDS-PAGE patterns of acid-solubilized collagen (ASC)
and pepsin-solubilized collagen (PSC) from the skin of sturgeon fish
(Huso huso). I, high molecular-weight protein marker
3.2 | SDS-PAGE analysis
3.3 | Amino acids analysis
Electrophoretic analysis of the collagen samples are presented in
Figure 1. The main components of ASC and PSC were two α-chains As shown in Table 1, similar amino acid compositions were ob-
(with an approximate α1 to α2 band intensity ratio of 2:1), β (dimers) tained for ASC and PSC. The major amino acid in both collagens
and small amounts of γ (trimers). Generally, collagen type I contains was glycine, followed by alanine, proline, and hydroxyproline con-
α1 and α2 chains and this is the main collagen present in fish skin, tent. Glycine is the most abundant amino acid of the three helical
bone, and scale. These patterns are consistent with the results re- α-chains and is situated uniformly at every third residue, except
ported for the skin of different fish species, including leatherjacket in the first 10 amino acid residues of the C-terminus and the first
(Ahmad et al., 2010), blacktip shark (Kittiphattanabawon, Benjakul, 14 amino acid residues of the N-terminus (Burghagen,  1999).
Visessanguan, & Shahidi, 2010), striped catfish (Singh et al., 2011), amur Elimination of some sections of the telopeptide regions during en-
sturgeon (Wang, Liang, Chen, et al., 2014), and tilapia (Li et al., 2018). zymatic hydrolysis can cause slight discrepancy in the amino acid
Based on the obtained results the inter- and intramolecular composition of both collagens.
cross-links of PSC were weaker than those of ASC; because by lim- Additionally, imino acids contents (proline and hydroxypro-
ited pepsin digestion, some of the β and γ-chains more likely cleaved line) have a major effect on the thermal resistance of collagens
into α-chains, which would lead to a slight decrease in molecular (Muyonga, Cole, & Duodu, 2004). Thus, denaturation tempera-
weight and increase in intensity of the α-chains band in PSC. These tures are decreased as the number of imino acid residues reduces
finding were in accordance with the studies for collagen skin from in collagen, and there is linear correlation between the degree
pacific cod (Sun, Li, Song, Si, & Hou, 2017), balloon fish (Huang, of cross-linking and hydroxyproline content (Ikoma, Kobayashi,
Shiau, Chen, & Huang, 2011), cobia (Zeng et al., 2012), and longbar- Tanaka, Walsh, & Mann, 2003). Hydroxyproline by forming in-
bel catfish (Zhang, Liu, & Li, 2009). terchain hydrogen bond plays a main role to the stabilization
ATEF et al. |
      5 of 10

TA B L E 1   Amino acid composition of sturgeon skin collagen 3.4 | Morphological characterization

(percentage of amino acid per 1 mg of dry mass of collagen)

PSC As shown in Figure 2, SEM images of the collagen samples displayed

Amino acids ASC (%) (%) a soft white and multilayered sponges with loose, fibrous, and po-
Aspartic acid/Asparagine 6.72 6.76 rous microstructures. The protein surfaces were partially wrinkled

Glutamic acid/Glutamine 10.88 10.53 due to dehydration during lyophilization, but the SEM microstruc-
ture showed irregular dense sheet-like filamentous meshwork. The
Hydroxyproline 7.76 8.03
images displayed nodular-like structures, which indicate the fibrous
Serine 5.04 5.17
nature of the collagens. The results confirmed that ASC and PSC had
Glycine 19.20 19.39
interconnected network with 20–120 µm pore size, which would
Histidine 0.58 0.49
support the use of fish collagens as carrier system for drugs and bio-
Arginine 9.60 9.37
active compounds. The morphological structures of collagens from
Threonine 2.84 2.89 other fish skins were fibrillar with interconnected network pore sizes
Alanine 9.32 9.46 of 80–250 µm (Veeruraj et al., 2013; Wang, Liang, Chen, et al., 2014).
Proline 13.38 13.55 Important parameters of scaffold microstructure for biomateri-
Tyrosine 0.79 0.53 als are pore size, porosity, and surface area. Therefore, the struc-
Valine 2.29 2.23 tural features of collagen sponge such as pore wall morphology, pore
Methionine 1.14 1.19 shape, and interconnectivity will perform a crucial role in matrices

Isoleucine 1.54 1.67 for cell proliferation, migration, mass transport, hydrating agents,
wound dressings, and other biomedical materials.
Leucine 2.48 2.47
Phenylalanine 2.58 2.36
Lysine 3.80 3.77
3.5 | Thermal behavior of the fish collagen
Cysteine 0.01 0.01
Tryptophan 0.07 0.10
Thermal denaturation (Td) is a major determinant of the thermal stabil-
Total 100 100
ity of proteins. Thermal denaturation of collagen causes breaking of the
Imino acid 21.14 21.58 triple helix and subsequent loss of the structural properties of collagen
(Bae et  al.,  2008). DSC (Figure  3) showed that thermal denaturation
amount of both samples (ASC: 29.34°C and PSC: 29.89°C) were similar
of the triple helical structure. The presence of pyrrolidine rings to the collagens from amur sturgeon (32.6°C) (Wang, Liang, Chen, et al.,
in imino acids strengthens the triple helix structure of colla- 2014), balloon fish (29.97°C) (Huang et al., 2011), catfish (31.85°C) (Zhang
gen and of the polypeptide chain conformation (Wong,  1989). et  al.,  2009), and bigeye snapper (30.4°C) (Jongjareonrak, Benjakul,
Total imino acid amounts (Table 1) of both collagens were sim- Visessanguan, & Tanaka, 2005), lower than those from other land animals
ilar to those of other fish skin but lower than the amount in collagens such as porcine skin (37°C) (Nagai, Araki, & Suzuki, 2002) and
mammalian collagens (Foegeding et al., 1996). Different values calf skin (37°C) (Ogawa et al., 2003) and also, higher than cold water fish
of proline and hydroxyproline in collagens across different spe- skin collagens, such as redfish (16.1°C) (Wang, An, Xin, Zhao, & Hu, 2007),
cies are mostly correlated to different living environments and cod (15°C) (Duan, Zhang, Du, Yao, & Konno, 2009), pacific whiting fish
habitat temperature. Also, the amounts of histidine, tyrosine, (21.7°C) (Kim & Park, 2004), and chum salmon (19°C) (Kimura & Ohno,
cysteine, and tryptophan (Table 1) were similar to findings on 1987). This difference can be due to differences in the habitat and body
other sources of collagen. This finding suggests that the stur- temperatures of the fish, and content of imino acids. This result showed
geon skin collagen is related to the type I class, considering a similar denaturation temperatures for both collagens; pepsin, without
the limited amount or absence of disulfide bonds in the protein destroying the triple helix structure of collagen, cleaved the telopeptide
(Owusu-Apenten, 2002). region containing the intramolecular cross-links.
Maximum absorbance of ASC and PSC by UV absorption spec- High heat transfer, because of the changes in some physical
tra occurred at 232 nm, which is similar to the results of other attributes, including light scattering, optical activity, viscosity, sed-
fish collagens such as catla and rohu (232 nm) (Pal, Nidheesh, & imentation, and diffusion, cause the decomposition of the collagen
Suresh, 2015), channel catfish (232 nm) (Liu, Li, & Guo, 2007), triple-helix structure (Usha & Ramasami,  2004). The second peak
and pacific cod (231 nm) (Sun et al., 2017). The maximum absor- of DSC thermogram mentions the melting temperature (Tmax) that
bance of proteins typically occurs at 280 nm (Edwards, Farwell, show the breakage of peptides chains in cross-linked collagen. The
Holder, & Lawson, 1997); however, due to the low amounts of Tmax peaks for ASC and PSC were observed at 92.03°C and 90.93°C,
aromatic amino acids (phenylalanine, tryptophan, and tyrosine), respectively, which are similar to values from previous studies on
no absorption peak was detected at 280  nm in ASC and PSC collagens derived from amur sturgeon fish skin (Wang, Liang, Chen,
(Table 1). et al., 2014).
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F I G U R E 2   Scanning electron microscopic structure images of ASC and PSC isolated from sturgeon fish skin

F I G U R E 3   DSC thermogram of ASC and PSC from the skin of

sturgeon fish F I G U R E 4   Fourier-transform infrared (FTIR) spectra of ASC and
PSC from the sturgeon fish skin

main absorption peaks consisted of five main amide bonds, includ-

3.6 | Fourier-transform infrared spectra ing amide A, amide B, and also amide I, II, and III that were similar
to the spectra of other collagen fish species (Kittiphattanabawon,
FTIR spectra of both lyophilized collagens were used to evaluate Benjakul, Visessanguan, Kishimura, & Shahidi,  2010; Singh
the secondary structures and functional groups (Figure  4). The et  al.,  2011; Sun et  al.,  2017; Wang, Liang, Chen, et  al., 2014;
ATEF et al. |
      7 of 10

Wang, Pei, Liu, & Zhou,  2018; Zhang et  al.,  2009). Absorption charge, observed at pH 5.4 and 5.0 for ASC and PSC, respectively,
characteristic of amide A for ASC and PSC were obtained at 3,328 were suggested to be the pI. The solubility of collagen due to the
and 3,323 cm–1, respectively; this is related to N–H stretching repulsion between the protein chains increase at pH values below
occurring in the range of 3,400–3,440  cm–1. The signal of N–H and/or above pI. Conversely, at net zero charge, there is increased
group changes to lower frequencies when involved in hydrogen hydrophobic interactions leading to aggregation and protein pre-
bonding in the peptide chain (Doyle, Bendit, & Blout, 1975). Our cipitation (Singh et al., 2011). Discrepancy in pI and zeta potential
study showed that the N–H groups of the sturgeon fish collagen across the pH values may be due to the content of acidic (glutamic
with carbonyl groups present in the polypeptide chains formed acid and aspartic acid) and basic amino acids (arginine, histidine, and
hydrogen bonds. Amide B peaks of ASC at 2,936 cm–1 and PSC lysine). The collagen of different fish skin showed different pI val-
–1
at 2,939 cm is associated with asymmetrical CH2 stretch (Abe & ues, for example, loach (6.42 and 6.51) (Wang et  al.,  2018), tilapia
Krimm, 1972). (6.42 and 6.82) (Chen et  al.,  2016), catfish (4.72 and 5.43) (Singh
The amide I region are usually applied to evaluate the secondary et al., 2011), and bamboo shark (6.21 and 6.56) (Kittiphattanabawon,
structure of proteins, mainly related to C=O group vibrations with Benjakul, Visessanguan, Kishimura, & Shahidi, 2010). The various pI
–1
frequencies of 1,600–1,700 cm (Muyonga et  al.,  2004; Payne & between different species can be due the difference in the amino
Veis, 1988). Amide I band of ASC and PSC were observed at 1,654 acid profile and sequences (Kaewdang, Benjakul, Kaewmanee, &
and 1,663 cm–1, respectively, which suggest that the formation of Kishimura, 2014).
hydrogen bonds between carbonyl group (C=O) and N–H group is
possibly responsible for the triple helical structure, as previously
suggested (Zanaboni, Rossi, Onana, & Tenni, 2000). The peak of 3.8 | Solubility of collagen
amide II for ASC and PSC was observed at 1,546 and 1,553  cm–1,
respectively, while the characteristic peak of amide III was located Effects of different NaCl concentrations and pH on the solubility
–1
at 1,239 cm for both collagens. Amide II and III band vibration of collagens are shown in Figure 6. Results revealed that ASC and
modes are commonly related to the NH bending and CN vibra-
tion, respectively (Barth & Zscherp, 2002). The intensity of amide
III and 1,450 cm–1 bands demonstrate the triple-helical structure of
collagen protein (Plepis, Goissis, & Das Gupta, 1996). Furthermore,
both collagens absorption peaks around 1,453–1,452  cm–1 corre-
spond to the pyrrolidine ring vibration of proline and hydroxyproline
(Muyonga et al., 2004).

3.7 | Surface charge of the sturgeon fish collagen

The zeta potential of both collagen samples at various pH is shown in

Figure 5. The samples showed net positive surface charge at pH 2–5,
but changed to negative charge at pH 6–11. The net zero surface

F I G U R E 6   Solubility of acid-solubilized collagen (ASC) and

F I G U R E 5   Zeta (ζ) potential of ASC and PSC from sturgeon fish pepsin-solubilized collagen (PSC) from sturgeon fish skin at
skin at different pH different pH (a) and NaCl concentrations (b)
 |
8 of 10       ATEF et al.
PSC were solubilized at pH 1 to 4, with the highest solubility at
pH 3 and minimum solubility at pH 7. However, a slight increase
in solubility was observed at alkaline pH (Figure 6a), as previously
reported for other fish collagens such as cobia (Zeng et al., 2012),
golden goatfish (Matmaroh, Benjakul, Prodpran, Encarnacion,
& Kishimura, 2011), and tilapia (Chen et al., 2016). When pH of
the sample is lower or higher than the pI, because of the repul-
sive forces between the chains (positive or negative charge of
proteins), the solubility increases (Vojdani,  1996). According to
Foegeding et al. (1996), pIs of collagen range from pH 6 to 9. In
addition, the solubility of ASC was lower than PSC at various pH
values, except at pH 4–5. This suggests that PSC possesses a lower
degree of cross-linking than ASC.
With increasing salt content up to 2%, solubility of ASC and
PSC remained stable (Figure 6b) but there was a sharp decrease
at 3% NaCl. The result of other studies on the skin of eel fish
(Veeruraj et al., 2013), cobia (Zeng et al., 2012) and bigeye snapper
(Jongjareonrak, Benjakul, Visessanguan, & Tanaka,  2005) showed
that the solubility of collagens decreased with increased NaCl con-
centration and that this phenomenon may be due to salting-out. On
this basis, the higher ionic strength of the solution, due to aggre-
gation of protein chains, competition of ionic salts for water, and
increased hydrophobic interaction, led to protein precipitation and
reduction in protein solubility (Damodaran, 1996). From this re-
sults, the solubility of PSC, due to partial hydrolysis of high molec-
ular weight proteins by pepsin, was better than ASC at higher NaCl
concentration.
4 |  CO N C LU S I O N S
In this study, sturgeon fish skin waste was demonstrated to be a
source of type I collagen. FTIR analyses of both collagens indicated
that the integrity of their triple helical structure was maintained
following extraction and the proteins displayed high solubility at
acidic pH. Porous and irregular microstructure and biocompatibil-
ity of the collagens showed their potential for use as biomaterials.
Therefore, future studies should focus on the application of the
collagen from sturgeon skin by-products as an alternative to mam-
malian collagens and also a valuable and environmentally friendly
material in cosmetic, biopharmaceutical, biomaterials, and food
applications.
AC K N OW L E D G M E N T S
Authors thank Dr. Abbas Zamani of the Fisheries Department at
University of Malayer, and Mrs. Maryam Khajavi of the Fisheries and
Environmental Science Faculty of Gorgan University for their valu-
able guidance and help with experimentations.
C O N FL I C T O F I N T E R E S T
The authors declare that there is no conflict of interest regarding the
publication of this article.
ORCID
Maryam Atef  https://orcid.org/0000-0002-2324-648X
Seyed Mahdi Ojagh  https://orcid.org/0000-0001-7325-1263
Ali Mohammad Latifi  https://orcid.org/0000-0002-8952-5174
Mina Esmaeili  https://orcid.org/0000-0001-9521-0550
Chibuike C. Udenigwe  https://orcid.org/0000-0001-8802-7707
REFERENCES
Abe, Y., & Krimm, S. (1972). Normal vibrations of crystalline poly-
glycine I. Biopolymers, 11, 1817–1839. https://doi.org/10.1002/
bip.1972.36011​0905
Ahmad, M., Benjakul, S., & Nalinanon, S. (2010). Compositional and
physicochemical characteristics of acid solubilized collagen ex-
tracted from the skin of unicorn leatherjacket (Aluterus monoceros).
Food Hydrocolloids, 24, 588–594. https://doi.org/10.1016/j.foodh​
yd.2010.03.001
Atef, M., & Ojagh, S. M. (2017). Health benefits and food applica-
tions of bioactive compounds from fish byproducts: A review.
Journal Functional Foods, 35, 673–681. https://doi.org/10.1016/j.
jff.2017.06.034
Bae, I., Osatomi, K., Yoshida, A., Osako, K., Yamaguchi, A., & Hara, K.
(2008). Biochemical properties of acid-soluble collagens extracted
from the skins of underutilised fishes. Food Chemistry, 108, 49–54.
https://doi.org/10.1016/j.foodc​hem.2007.10.039
Barth, A., & Zscherp, C. (2002). What vibrations tell us about pro-
teins? Quarterly Reviews of Biophysics, 35, 369–430. https://doi.
org/10.1017/S0033​58350​2003815
Bi, C., Li, X., Xin, Q., Han, W., Shi, C., Guo, R., … Zhong, J. (2019). Effect
of extraction methods on the preparation of electrospun/electro-
sprayed microstructures of tilapia skin collagen. Journal of Bioscience
and Bioengineering., 128, 234–240. https://doi.org/10.1016/j.
jbiosc.2019.02.004
Burghagen, M. (1999). Collagen. In H. D. Belitz & W. Grosch (Eds.), Food
chemistry (2nd ed., pp. 540–547). Berlin, Germany: Springer-Verlag
Berlin Heidelberg.
Chen, J., Li, L., Yi, R., Xu, N., Gao, R., & Hong, B. (2016). Extraction and
characterization of acid-soluble collagen from scales and skin of ti-
lapia (Oreochromis niloticus). LWT-Food Science and Technology, 66,
453–459. https://doi.org/10.1016/j.lwt.2015.10.070
Damodaran, S. (1996). Amino acids, peptides, and proteins. In O. R.
Fennema (Ed.), Food chemistry (3rd ed., pp. 321–429). New York, NY:
Marcel Dekker.
Doyle, B. B., Bendit, E. G., & Blout, E. R. (1975). Infrared spectroscopy
of collagen and collagen-like polypeptides. Biopolymers, 14, 937–957.
https://doi.org/10.1002/bip.1975.36014​0505
Duan, R., Zhang, J., Du, X., Yao, X., & Konno, K. (2009). Properties of colla-
gen from skin, scale and bone of carp (Cyprinus carpio). Food Chemistry,
112, 702–706. https://doi.org/10.1016/j.foodc​hem.2008.06.020
Edwards, H. G. M., Farwell, D. W., Holder, J. M., & Lawson, E. E. (1997).
Fourier-transform Raman spectroscopy of ivory: II. Spectroscopic
analysis and assignments. Journal of Molecular Structure, 435, 49–58.
https://doi.org/10.1016/S0022​-2860(97)00122​-1
Foegeding, E. A., Lanier, T. C., & Hultin, H. O. (1996). Collagen. In O. R.
Fennema (Ed.), Food chemistry (3rd ed., pp. 902–906). New York, NY:
Marcel Dekker.
Huang, Y. R., Shiau, C. Y., Chen, H. H., & Huang, B. C. (2011). Isolation
and characterization of acid and pepsin-solubilized collagens from
the skin of balloon fish (Diodon holocanthus). Food Hydrocolloids, 25,
1507–1513. https://doi.org/10.1016/j.foodh​yd.2011.02.011
Ikoma, T., Kobayashi, H., Tanaka, J., Walsh, D., & Mann, S. (2003). Physical
properties of type I collagen extracted from fish scales of Pagrus
major and Oreochromis niloticas. International Journal of Biological
ATEF et al.
Macromolecules, 32, 199–204. https://doi.org/10.1016/S0141​
-8130(03)00054​- 0
Jongjareonrak, A., Benjakul, S., Visessanguan, W., Nagai, T., & Tanaka,
M. (2005). Isolation and characterisation of acid and pepsin-solu-
bilised collagens from the skin of brown stripe red snapper (Lutjanus
vitta). Food Chemistry, 93, 475–484. https://doi.org/10.1016/j.foodc​
hem.2004.10.026
Jongjareonrak, A., Benjakul, S., Visessanguan, W., & Tanaka, M. (2005).
Isolation and characterization of collagen from bigeye snapper
(Priacanthus macracanthus) skin. Journal of the Science of Food and
Agriculture, 85, 1203–1210. https://doi.org/10.1002/jsfa.2072
Kaewdang, O., Benjakul, S., Kaewmanee, T., & Kishimura, H. (2014).
Characteristics of collagens from the swim bladders of yellowfin
tuna (Thunnus albacares). Food Chemistry, 155, 264–270. https://doi.
org/10.1016/j.foodc​hem.2014.01.076
Kim, J. S., & Park, J. W. (2004). Characterization of acid-soluble collagen
from Pacific whiting surimi processing by-products. Journal of Food
Science, 69, 637–642. https://doi.org/10.1111/j.1365-2621.2004.
tb099​12.x
Kimura, S., & Ohno, Y. (1987). Fish type-I collagen—Tissue-specific exis-
tence of 2 molecular-forms, (alpha-1)2-alpha-2 and alpha-1-alpha-3,
in Alaska Pollack. Biochemistry & Molecular Biology, 88, 409–413.
https://doi.org/10.1016/0305-0491(87)90320​-8
Kittiphattanabawon, P., Benjakul, S., Visessanguan, W., Kishimura, H.,
& Shahidi, F. (2010). Isolation and characterisation of collagen from
the skin of brownbanded bamboo shark (Chiloscyllium punctatum).
Food Chemistry, 119, 1519–1526. https://doi.org/10.1016/j.foodc​
hem.2009.09.037
Kittiphattanabawon, P., Benjakul, S., Visessanguan, W., Nagai, T., &
Tanaka, M. (2005). Characterisation of acid-soluble collagen from skin
and bone of bigeye snapper (Priacanthus tayenus). Food Chemistry, 89,
363–372. https://doi.org/10.1016/j.foodc​hem.2004.02.042
Kittiphattanabawon, P., Benjakul, S., Visessanguan, W., & Shahidi, F.
(2010). Isolation and properties of acid-and pepsin-soluble collagen
from the skin of blacktip shark (Carcharhinus limbatus). European Food
Research and Technology, 230, 475–483.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assem-
bly of the head of bacteriophage T4. Nature, 15, 680–685. https://
doi.org/10.1038/227680a0
Li, J., Wang, M., Qiao, Y., Tian, Y., Liu, J., Qin, S., & Wu, W. (2018).
Extraction and characterization of type I collagen from skin of tila-
pia (Oreochromis niloticus) and its potential application in biomedi-
cal scaffold material for tissue engineering. Process Biochemistry, 74,
156–163. https://doi.org/10.1016/j.procb​io.2018.07.009
Liu, H., Li, D., & Guo, S. (2007). Studies on collagen from the skin of chan-
nel catfish (Ictalurus punctaus). Food Chemistry, 101(2), 621–625.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951).
Protein measurement with Folin phenol reagent. Journal of Biological
Chemistry, 193, 256–275.
Matmaroh, K., Benjakul, S., Prodpran, T., Encarnacion, A. B., & Kishimura,
H. (2011). Characteristics of acid soluble collagen and pepsin soluble
collagen from scale of spotted golden goatfish (Parupeneus hepta-
canthus). Food Chemistry, 129, 1179–1186. https://doi.org/10.1016/j.
foodc​hem.2011.05.099
Mohan, A., & Udenigwe, C. C. (2015). Towards the design of hypolipidae-
mic peptides: Deoxycholate binding affinity of hydrophobic peptide
aggregates of casein plastein. Journal of Functional Foods, 18, 129–
136. https://doi.org/10.1016/j.jff.2015.06.064
Montero, P., Jimennez-Colmenero, F., & Borderias, J. (1991). Effect of
pH and the presence of NaCl on some hydration properties of col-
lagenous material from trout (Salmo irideus Gibb) muscle and skin.
Journal of the Science of Food and Agriculture, 54, 137–146. https://
doi.org/10.1002/jsfa.27405​4 0115
Muyonga, J. H., Cole, G. B., & Duodu, K. G. (2004). Characterisation of
acid soluble collagen from skins of young and adult Nile perch (Lates
|
      9 of 10
niloticus). Food Chemistry, 85, 81–89. https://doi.org/10.1016/j.foodc​
hem.2003.06.006
Nagai, T., Araki, Y., & Suzuki, N. (2002). Collagen of the skin of ocellate
pufferfish (Takifugu rubripes). Food Chemistry, 78, 173–177. https://
doi.org/10.1016/S0308​-8146(01)00396​-X
Ogawa, M., Moody, M. W., Portier, R. J., Bell, J., Schexnayder, M. A.,
& Losso, J. N. (2003). Biochemical properties of black drum and
sheeps head sea bream skin collagen. Journal of Agricultural and Food
Chemistry, 51, 8088–8092. https://doi.org/10.1021/jf034​350r
Ovissipour, M., Abedian, A., Motamedzadegan, A., Rasco, B., Safari, R.,
& Shahiri, H. (2009). The effect of enzymatic hydrolysis time and
temperature on the properties of protein hydrolysates from Persian
sturgeon (Acipenser persicus) viscera. Food Chemistry, 115, 238–242.
https://doi.org/10.1016/j.foodc​hem.2008.12.013
Owusu-Apenten, R. K. (2002). Food protein analysis. New York, NY:
Marcel Dekker, Inc.
Pal, G. K., Nidheesh, T., & Suresh, P. V. (2015). Comparative study on
characteristics and in vitro fibril formation ability of acid and pep-
sin soluble collagen from the skin of catla (Catla catla) and rohu
(Labeo rohita). Food Research International, 76, 804–812. https://doi.
org/10.1016/j.foodr​es.2015.07.018
Payne, K. J., & Veis, A. (1988). Fourier transform IR spectroscopy of
collagen and gelatin solutions: Deconvolution of the amide I band
for conformational studies. Biopolymers, 27, 1749–1760. https://doi.
org/10.1002/bip.36027​1105
Plepis, D. G., Goissis, G., & Das Gupta, D. K. (1996). Dielectric and pyroelec-
tric characterization of anionic and native collagen. Polymer Engineering
& Science, 36, 2932–2938. https://doi.org/10.1002/pen.10694
Singh, P., Benjakul, S., Maqsood, S., & Kishimura, H. (2011). Isolation and
characterisation of collagen extracted from the skin of striped cat-
fish (Pangasianodon hypophthalmus). Food Chemistry, 124, 97–105.
https://doi.org/10.1016/j.foodc​hem.2010.05.111
Subhan, F., Ikram, M., Shehzad, A., & Ghafoor, A. (2015). Marine colla-
gen: An emerging player in biomedical applications. Journal of Food
Science and Technology, 52, 4703–4707. https://doi.org/10.1007/
s1319​7-014-1652-8
Sun, L., Li, B., Song, W., Si, L., & Hou, H. (2017). Characterization of
Pacific cod (Gadus macrocephalus) skin collagen and fabrication
of collagen sponge as a good biocompatible biomedical material.
Process Biochemistry, 63, 229–235. https://doi.org/10.1016/j.procb​
io.2017.08.003
Tamilmozhi, S., Veeruraj, A., & Arumugam, M. (2013). Isolation and char-
acterization of acid and pepsin-solubilized collagen from the skin
of sailfish (Istiophorus platypterus). Food Research International, 54,
1499–1505. https://doi.org/10.1016/j.foodr​es.2013.10.002
Usha, R., & Ramasami, T. (2004). The effects of urea and n-propanol on
collagen denaturation: Using DSC, circular dicroism and viscosity.
Thermochimica Acta, 409, 201–206. https://doi.org/10.1016/S0040​
-6031(03)00335​-6
Veeruraj, A., Arumugam, M., & Balasubramanian, T. (2013). Isolation and
characterization of thermostable collagen from the marine eel-fish
(Evenchelys macrura). Process Biochemistry, 48, 1592–1602. https://
doi.org/10.1016/j.procb​io.2013.07.011
Vojdani, F. (1996). Solubility. In G. M. Hall (Ed.), Methods of testing pro-
tein functionality (1st ed., pp. 11e60) Great Britain: St Edmundsbury
Press.
Wang, H., Liang, Y., Wang, H., Zhang, H., Wang, M., & Liu, L. (2014).
Physical-chemical properties of collagens from skin, scale, and bone
of grass carp (Ctenopharyngodon idellus). Journal of Aquatic Food
Product Technology, 23, 264–277.
Wang, J., Pei, X., Liu, H., & Zhou, D. (2018). Extraction and character-
ization of acid-soluble and pepsin-soluble collagen from skin of
loach (Misgurnus anguillicaudatus). International Journal of Biological
Macromolecules, 106, 544–550. https://doi.org/10.1016/j.ijbio​
mac.2017.08.046
 |
10 of 10       ATEF et al.
Wang, L., An, X., Xin, Z., Zhao, L., & Hu, Q. (2007). Isolation and char-
acterization of collagen from the skin of deep-sea redfish (Sebastes
mentella). Journal of Food Science, 72, 450–455. https://doi.
org/10.1111/j.1750-3841.2007.00478.x
Wang, L., An, X., Yang, F., Xin, Z., Zhao, L., & Hu, Q. (2008). Isolation
and characterisation of collagens from the skin, scale and bone of
deep-sea redfish (Sebastes mentella). Food Chemistry, 108, 616–623.
https://doi.org/10.1016/j.foodc​hem.2007.11.017
Wang, L., Liang, Q., Chen, T., Wang, Z., Xu, J., & Ma, H. (2014).
Characterization of collagen from the skin of Amur sturgeon
(Acipenser schrenckii). Food Hydrocolloids, 38, 104–109. https://doi.
org/10.1016/j.foodh​yd.2013.12.002
Wong, D. W. S. (1989). Mechanism and theory in food chemistry. New York,
NY: Van Nostrand Reinhold.
Zanaboni, G., Rossi, A., Onana, A. M. T., & Tenni, U. R. (2000). Stability
and networks of hydrogen bonds of the collagen triple helical
structure: Influence of pH and chaotropic nature of three an-
ions. Matrix Biology, 19, 511–520. https://doi.org/10.1016/S0945​
-053X(00)00096​-2
Zeng, S., Yin, J., Yang, S., Zhang, C., Yang, P., & Wu, W. (2012). Structure
and characteristics of acid and pepsin-solubilized collagens from the
skin of cobia (Rachycentron canadum). Food Chemistry, 135, 1975–
1984. https://doi.org/10.1016/j.foodc​hem.2012.06.086
Zhang, M., Liu, W., & Li, G. (2009). Isolation and characterisation of
collagens from the skin of largefin longbarbel catfish (Mystus mac-
ropterus). Food Chemistry, 115, 826–831. https://doi.org/10.1016/j.
foodc​hem.2009.01.006
Zhang, X., Ookawa, M., Tan, Y., Ura, K., Adachi, S., & Takagi, Y. (2014).
Biochemical characterisation and assessment of fibril-forming ability
of collagens extracted from Bester sturgeon Huso huso × Acipenser
ruthenus. Food Chemistry, 160, 305–312. https://doi.org/10.1016/
j.foodc​hem.2014.03.075
How to cite this article: Atef M, Ojagh SM, Latifi AM,
Esmaeili M, Udenigwe CC. Biochemical and structural
characterization of sturgeon fish skin collagen (Huso huso). J
Food Biochem. 2020;00:e13256. https://doi.org/10.1111/
jfbc.13256