Biocatalysis and Agricultural Biotechnology 38 (2021) 102218

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Biocatalysis and Agricultural Biotechnology

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Effect of ultrasound-assisted extraction on the extractability and

physicochemical properties of acid and pepsin soluble collagen
derived from Sharpnose stingray (Dasyatis zugei) skin
Mannur Ismail Shaik, Jia Ying Chong, Norizah Mhd Sarbon ∗
Faculty of Fisheries and Food Science, Universiti Malaysia Terengganu, 21030, Kuala Nerus, Terengganu, Malaysia

ARTICLE INFO ABSTRACT

Keywords: This study aims to investigate the effect of ultrasound-assisted extraction on the extractability
Fish collagen and physico-chemical properties of acid (UASC) and pepsin (UPSC) soluble collagen derived
Sharpnose stingray from Sharpnose stingray (Dasyatis zugei) skin. Pre-treated skins were extracted using 0.5M acetic
Ultrasound-assisted extraction acid under ultrasonication for UASC, and 0.5M acetic acid with 1.5% (w/w) pepsin under ultra-
Yield
sonication for UPSC. The yield of UPSC (61.50 ± 0.79%) was higher than UASC
Physicochemical properties
(42.34 ± 0.62%) while, no significant different in chemical compositions except for protein con-
tent between UASC and UPSC. The enzymatic hydrolysis results in the molecular weight of α-2
chain for UPSC. Freeze dried UASC and UPSC had similar morphological structures, with loose,
irregular dense sheet-like film and irregular pore size. The extracted UASC and UPSC were ther-
mally stable, with high thermal denaturation (Td), and had no significant differences in their
maximum temperature (Tmax). UASC and UPSC also showed highly solubilized at acidic pH range
and had the lowest solubility at pH 8. In conclusion, ultrasound-assisted extraction showed a po-
tential technique to improve the yields of collagen extracted without affecting other properties of
Sharpnose stingray collagen.

1. Introduction
Protein fibrous collagen, which found primarily in multicellular organisms, is the major structural component of the skin, bones,
tendons, cartilage, blood vessels and teeth (Zhang et al., 2016; Baderi and Sarbon, 2019; Sukeri et al., 2021). It is typically extracted
from land-living animals. However, due to diseases such as bovine spongiform encephalopathy (BSE) and religious limitations for us-
ing bovine and porcine-derived collagen, researchers have investigated extracting collagen from fish processing by-products, which
are usually discarded as waste (Hadfi and Sarbon, 2019; HukmiSarbon, 2018; Seixas et al., 2020). Marine collagens are safer to con-
sume and have no religious issues (El-rashidy et al., 2015). The development of high-value food ingredients from fish processing by-
products will enhance the sustainability of the industry (Luo et al., 2020). The collagen extractability may be characterized by the to-
tal production of extracted collagen. Collagen extracted from fish by-products has been used in the food, biomedical, pharmaceutical,
and cosmetic industries because of its properties such as gelling and surface properties, excellent biocompatibility, and other biologi-
cal characteristics (Alves et al., 2017; Peres et al., 2017; Sulaiman and Sarbon, 2020).
The amount of hydroxyproline in the extracted collagen can indicate collagen extractability (Zhou et al., 2016). Collagen can be
extracted using various extraction methods such as acid, pepsin, sodium chloride or homogenization-aided extraction and also for us-

∗ Corresponding author.
E-mail address: norizah@umt.edu.my (N.M. Sarbon).

https://doi.org/10.1016/j.bcab.2021.102218
Received 29 May 2021; Received in revised form 31 October 2021; Accepted 9 November 2021
Available online 12 November 2021
1878-8181/© 2021 Elsevier Ltd. All rights reserved.
M.I. Shaik et al. Biocatalysis and Agricultural Biotechnology 38 (2021) 102218

ing CO2 acidified water, green process (Tan and Chang, 2018; Hamdan and Sarbon, 2019; Sousa et al., 2020). Of these methods, acid
and pepsin extraction method are most commonly used. Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) have been
used to successfully extract and characterize sharpnose stingray skin (Ong et al., 2021). The yield from pepsin extraction was higher
than from acid extraction because the pepsin cleaved the cross-links at telopeptide region, which increases collagen solubility in acid
and hence increases extractability (Veeruraj et al., 2015; Sousa et al., 2019). Recent studies showed that extracting ASC and PSC us-
ing an ultrasound treatment shortened extraction time and increases the yield of extracted collagen.
Ultrasound is a high-frequency sound wave in excess of human hearing limits (about 20 kHz). The difference in sound pressure is
directly proportional to the how much heat is applied (Zou et al., 2017). There has been study on collagen extraction using both acid
and pepsin extraction methods under ultrasonication (Ali et al., 2018). Furthermore, in comparison to conventional methods, the ul-
tra-sonication technique is non-invasive and non-destructive (Akram and Zhang, 2020). By using ultrasonic processing characteris-
tics, the functional properties of proteins have been improved because of the cavitation force of ultrasonic waves (Akram and Zhang,
2020). The propagation of ultrasound caused compression and decompression of the particles in biological materials and producing a
protein with better quality (Zou et al., 2017).
Characterizing the physico-chemical properties of extracted collagen is essential to determine the quality of extracted collagen.
The chemical properties of marine collagen commonly measured include proximate analysis, protein concentration, amino acid com-
position and zeta-potential (HukmiSarbon, 2018; Wang et al., 2018). The proximate analysis of the extracted collagen is slightly dif-
ferent from other food samples, as collagen is a fibrous protein, and therefore the content of carbohydrate is negligible. The physical
properties of the collagen include structural properties, morphological properties, protein patterns, solubility, viscosity and thermal
stability (Veeruraj et al., 2015).
Stingrays are fishes that are wide and the fins are broad and run the full length of their bodies, giving them a flat, round shape
(Almeida et al., 2010). The chemical composition of Sharpnose stingray skin were highest in moisture (78.0%), followed by protein
(31.3%), fat (1.6%) and lastly ash (0.2%) (Ong et al., 2021). They have cartilage rather than bones. During fish processing, solid
waste such as stingray skin generated. This waste is potential source of collagen (Chi et al., 2013; Ong et al., 2021). Stingray fishes are
easily available in Terengganu state of peninsular Malaysia and large amount of waste was generating from the processing (Bachok et
al., 2004). To make effective use of waste from fish processing, ultrasound assisted extraction could help to obtain a higher yield in a
shorter time. It may also affect the physico-chemical properties of the extracted collagen. Studies on the stingray species are very lim-
ited and the yield of collagen was high in the same species. Therefore, the objective of current study was designed to isolate acid solu-
ble (UASC) and pepsin soluble (UPSC) stingray skin collagen using ultrasound-assisted extraction method and also to characterize the
physico-chemical properties of UASC and UPSC.

2. Materials and methods

2.1. Materials
Sharpnose stingray (Dasyatis Zugei) was obtained from a local market in Kuala Terengganu, Malaysia. The average size of stingray
used in this study was about 20–25 cm long with average weight of 500 g–850 g. It was kept on ice and transported to a laboratory at
Universiti Malaysia Terengganu for further processing. Enzyme pepsin (EC 3.4.23.1) derived from porcine gastric mucosa and all the
chemicals were purchased from Sigma-Aldrich, Malaysia. The chemicals used in the extraction and analysis of the collagen were of
analytical grade.

2.2. Methods
2.2.1. Sample preparation
The Sharpnose stingrays were cleaned by removing the internal organs and rinsing with water. The skins were filleted manually
and washed with abundant water before storage at −80 °C for further use.

2.2.2. Sample pre-treatment

The pre-treatment method was conducted before the isolation of collagen in order to completely remove the undesirable residues.
The residues including non-collagenous proteins and minerals were removed from the sample, to prevent any interference during ex-
traction and analysis (Ong et al., 2021).

2.2.2.1. Removal of non-collagenous proteins. To remove non-collagenous proteins, the pre-treated sample was soaked in 0.1M sodium
hydroxide (NaOH) solution for 6 h at ratio of 1:8 (w/v). The NaOH solution was changed every 3 h. After treatment with alkali, each
sample was washed with cold distilled water (4–5 °C) until the rinsed water become neutral, as measured with a pH meter (Orion Star
A221, Thermo Scientific, Beverly, England). The entire pretreatment was done at 4 °C.

2.2.2.2. Demineralizing. Each sample was demineralized by continuous stirring using 0.5M EDTA for 24 h with a skin to EDTA solu-
tion ratio of 1:20 at pH 7.4. The solution was changed at every 12 h, then rinsed three times with distilled water (Ong et al., 2021).

2.2.3. Collagen extraction

2.2.3.1. Ultrasound-assisted extraction acid soluble collagen (UASC). Each sample (60 g) was soaked in 0.5M acetic acid with a
sample/solution ratio of 1:50 (w/v) and subjected to ultrasound treatment (Ultrasound Processor - Model CV334, Cole-Parmer,
USA) at 20 KHz and 750 W for 30 min. To avoid overheating, the ultrasound was operated in pulse mode for 5 s before 5 s
resting time. A temperature-controlled steel jacket was used in the ultrasonic processor to maintain the temperature at 4 °C. The

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M.I. Shaik et al. Biocatalysis and Agricultural Biotechnology 38 (2021) 102218

extracts were centrifuged at 6500×g for 15 min at 4 °C and the supernatants were separated. The residues were re-extracted
with 0.5M acetic acid with a sample/solution ratio of 1:10 (w/v) via an ultrasound processor at 20 KHz and 750 W for 30 min
before being centrifuged at 6500×g for 15 min. Both supernatants were combined and salted out by adding NaCl to give a final
concentration of 0.7M. The supernatants were centrifuged at 5000×g for 10 min at 4 °C and the precipitate were frozen at
−80 °C overnight before being freeze-dried (Labcocon, Kansas City, USA) (Ali et al., 2018).

2.2.3.2. Ultrasound-assisted extraction pepsin soluble collagen (UPSC). Each sample (60 g) was soaked in 0.5M acetic acid with a
sample/solution ratio of 1:40 (w/v) that contained 1.5% (w/v) pepsin and subjected to ultrasound treatment by an ultrasound
processor at 20 KHz and 750 W for 30 min. To avoid overheating, the ultrasound was operated in a pulse mode for 5 s, acting
before 5 s resting time. A temperature-controlled steel jacket was used in the ultrasonic processor to maintain the temperature at
4 °C. The extracts were centrifuged at 6500×g for 15 min at 4 °C and the supernatants were separated. The residues were re-
extracted with 0.5M acetic acid with a sample/solution ratio of 1:10 (w/v) containing 1.5% (w/v) pepsin via an ultrasound
processor at 20 KHz and 750 W for 30 min. Then, the extracts were centrifuged at 6500×g for 15 min. Both supernatants were
combined and salted out by adding NaCl to reach a final concentration of 0.7M. The supernatants were centrifuged at 5000×g
for 10 min and the precipitates were frozen at −80 °C °C.°C°C overnight before being freeze-dried (Labcocon, Kansas City, USA).
Collagen yield was calculated using the following formula:

2.2.4. Chemical properties of extracted collagen

2.2.4.1. Chemical composition. Moisture, protein, fat and ash of extracted fish collagen was determined following AOAC methods
(AOAC, 2000).

2.2.5. Physical properties of extracted collagen

2.2.5.1. Electrophoretic patterns. The electrophoretic patterns of the extracted collagens were analyzed using sodium dodecyl sul-
phate-polyacrylamide gel (SDS-PAGE). SDS-PAGE was performed on 7.5% polyacrylamide gel containing 5% of SDS. The con-
centration of the collagen is 1 μg/ml. The concentration of the collagen is 1 μg/ml. Each collagen samples were dissolved in 5%
of SDS solution and the mixture was heated at 85 °C for 5 min, centrifuged and then loaded onto the gel (20 μl). A loading
buffer made up of deionized water, 13% of 0.5 M Tris-HCl pH 6.8, 26% glycerol and 10% SDS was added to sample. The initial
running voltage was 60V for 15 min, followed by 150V for 1 h. The gels were stained using Bio-Safe Coomassie G-250 Stain for
1 h and then destained by stirring in distilled water for 2 h. The migration distance on the finished (stained) gel was used to
calculate the molecular weight. Precise Plus Protein Standard (Bio-Rad) was used as the standard (Rasli and Sarbon, 2019).

2.2.5.2. Morphological properties. The morphological properties of the extracted collagen were characterized using a scanning elec-
tron microscope (SEM) (JOEL JSM-6360 LA, Tokyo, Japan) at an accelerating voltage of 5.0 kV. The collagen powder was mounted
onto aluminum slugs (5 mm × 12.5 mm) and the sample collagen was sputter-coated with an auto fine coater (JOEL JFC 1600,
Tokyo, Japan). The sample collagens were observed at the magnification of 400× and 1000x (HukmiSarbon, 2018).

2.2.5.3. Structural properties. Fourier Transform Infrared Spectroscopy (FTIR) (Nicolet, Thermo Electron, USA) was used to study
the structural properties of extracted collagen. The infrared spectra of the lyophilized collagen were measured at 4000-400 cm−1
in a FTIR spectrophotometer. Each sample was prepared by mixing the freeze-dried collagen (10 mg) with potassium bromide
(KBr) at a ratio of 1:100 and then pressing it into a translucent pellet using a hydraulic press. The scanning was performed at a
resolution of 4 cm−1 and the FTIR spectra underwent an average of 32 scans. The functional group and mode of vibrations were
identified based on the peak of interest at a specific wavelength and absorbance (Hamdan and Sarbon, 2019).

2.2.5.4. Thermal stability. The thermal stability of the collagen samples was characterized using a differential scanning calorimeter
(DSC) (Perkin Elmer, Norwalk, CT, USA). The DSC was calibrated with indium of 99.99% purity and with a maximum peak tempera-
ture, Tmax of 156.22 °C and heat of fusion (total melting enthalpy), ΔH of 28.39 J/g. The collagen powder was rehydrated by adding
deionized water at a solid to solution ratio of 1:40 (w/v). Primarily, 5 mg of each sample was weighed accurately in an aluminum
volatile sample pan and then hermetically sealed. An empty pan was used as reference. The curve was recorded at a scanning rate of
1 °C/min from 15 to 60 °C. The Tmax was estimated from the thermogram obtained (Kaewdang et al., 2014).

2.2.5.5. Viscosity. The viscosity of the extracted collagen was conducted using a viscometer (Brookfield DV-1 viscometer, Illinois,
USA). The sample was prepared by dissolving 3 g of lyophilized collagen in 0.1M acetic acid. The solution was placed onto the
sample holder of the viscometer by using spindle No. 1 at speed 100 rpm. Each solution was heated from 4 to 50 °C with a heating
rate of 4.°C/°C//min. At each chosen temperature, the solution was held for 30 min for viscosity measurement. The relative viscosi-
ties at each temperature were calculated and compared with the viscosity of the extracted collagen at 4 °C (Zou et al., 2017).

2.2.5.6. Solubility. The solubility of extracted collagen was determined following the method of Zou et al. (2017) with some modifi-
cation of the pH range. Collagen samples (10 mg) were dissolved in 10 ml of 0.5M acetic acid with gentle stirring for 12 h to obtain
final concentration of 1 mg/ml and the pH was adjusted to 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 using 1 mol/L HCl or 1 mol/L NaOH. Then,
each mixture was stirred at room temperature for 30 min. The solutions were added with distilled water to reach 10 ml. The solu-

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M.I. Shaik et al. Biocatalysis and Agricultural Biotechnology 38 (2021) 102218

tions were then stirred for 30 min at 4 °C and then centrifuged at 6000×g for 30 min at 4°C°C. Protein concentrations in the super-
natant were measured using Lowry's method (Lowry et al., 1951), with bovine serum albumin (BSA) used to obtain a protein stan-
dard curve. A standard calibration curve was plotted by absorbance against protein concentration. Relative protein solubility was de-
termined by comparing the concentration of protein at each pH to the highest protein content as calculated using the following for-
mula:

2.2.6. Statistical analysis

All analyses were carried out in triplicate and the results obtained were presented in terms of mean ± standard deviation. The re-
sults were analyzed using independent t-test in IBM SPSS Statistics Version 25 (SPSS Inc., Chicago, IL, USA). The comparisons of
means were performed by Fisher's Test with a confidence level of p < 0.05.

3. Results and discussions

3.1. Yield
The yield of Ultrasound-assisted extraction acid soluble collagen (UASC) and ultrasound-assisted extraction pepsin soluble colla-
gen (UPSC) of Sharpnose stingray skin collagen were tested. There was a significant difference (p < 0.05) between the yield of UASC
(42.34 ± 0.62%) and UPSC (61.50 ± 0.79%). The ultrasound-assisted collagen extraction yield of UASC (48.37%) and UPSC
(56.65%) of Sharpnose stingray skin was higher than compared to without ultrasound-assisted collagen extraction yield of ASC
(20.48 ± 4.41%) and PSC (34.84 ± 1.26%) as reported by Ong et al. (2021). The highest yield of UPSC compared to UASC was due
to the cleavage of cross-linkage at telopeptide region of UPSC by pepsin without affecting the integrity of the triple helix structure
(Nalinanon et al., 2007). The lower degree of hydrolysis of UASC was due to the presence of inter-molecular cross-linking, as acids
were unable to break down this, leading to a decrease in solubility of collagen molecules under acidic conditions (Kaewdang et al.,
2014). The employment of ultrasound resulted in a higher yield due to the ability of ultrasound treatment to increase the kinetic en-
ergy of the particle substance through the shock effect of ultrasound.
Ultrasound treatment can enhance the yield of collagen compared to acetic acid treatment alone. The similar study carried out by
Kim et al. (2012) reported that yield of collagen extracted from sea bass skin with 0.5 M acetic acid solution and ultrasonic treatment
rapidly increased when compared with the skins treated in acid without ultrasonic treatment. Moreover, yield of collagen from the
golden carp skin, enhanced with the addition of higher concentration of pepsin for ultrasonic treatment (Ali et al., 2018). The ultra-
sonication increased the collagen content (16.3%) in UASC when compared to ASC of soft-shelled turtle (Pelodiscus sinensis) calipash,
which might be due to its cavitation and mechanical effects (Zou et al., 2017). Ultrasonic waves are known to replicate compression
and expansion, which lead to molecular motion and create cavitation bubbles. During the bubble generation and explosion process an
instantaneous increase in temperature (>5000 K) and pressure (over 1000 atm) can also lead to a large shear force wave and turbu-
lence within the cavitation area (Kim et al., 2013).
Traditionally used acetic acid could effectively penetrate through the skin and able to extract collagen more potentially. Pepsin
well known to increase the extraction of collagen cleavage of telopeptide region of tropocollagen and ultrasound technology gener-
ates the mechanical effect, resulting in the molecular motion of the solution. This combined effect facilitated the penetration of
pepsin and simultaneously enhanced mass transfer from the matrix of the skin. Therefore, the yield of collagen becomes higher in the
combinational approach of acetic acid, pepsin and ultrasound treatment.
The highest yield of the UPSC compared to the UASC from Sharpnose stingray skin was consistent with the findings of Ali et al.
(2018), who found levels in the skin of golden carps of 51.27% and 59.81% for UASC and UPSC, respectively. This indicates that dur-
ing hydrolysis, pepsin cleaved some cross-linked molecules at the telopeptide region without damaging the integrity of the triple he-
lix, thus, leading to a higher yield for PSC (Heu et al., 2010). In comparison, a study by Ong et al. (2021) showed that the extraction
yields of stingray skin collagen using acid (ASC) (20.48 ± 4.41%) and pepsin (PSC) (34.84 ± 1.26%), were lower than UASC and
UPSC in this current study. This finding proves that the ultrasound assisted extraction method improved the yield of extraction two-
fold due to cavitation effects of ultrasound in loosening the pretreated skin and permitting more of the acetic acid and pepsin to pene-
trate the skin and extract collagen more effectively (Ali et al., 2018).

3.2. Chemical properties

3.2.1. Chemical composition
The chemical compositions of the extracted collagens from the skin of Sharpnose stingray by using various extraction methods dis-
played in Table 1. In terms of moisture content, there were no significant differences between the extracted collagen UASC and UPSC.
These findings are lower than the moisture content of collagen from skin of brownbanded bamboo shark which were 7.77% and
8.09% respectively, for ASC and PSC as reported by Kittiphattanabawon et al. (2010). Moreover, our findings were similar to the find-
ings reported by Wang et al. (2013) on the collagen from the scales of Croceine croaker, for which the moisture content for ASC
(4.52 ± 0.54%) was higher than PSC (3.76 ± 0.54%).
The protein content of extracted UASC (41.77 ± 0.47%) was significantly different (p < 0.05) from the UPSC (36.64 ± 0.08%)
derived from Sharpnose stingray skin collagen (Table 1). However, the protein levels obtained from this study were lower than those
observed in a study by Kittiphattanabawon et al. (2010). They found that the protein levels of ASC and PSC for the skin of brown-

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M.I. Shaik et al. Biocatalysis and Agricultural Biotechnology 38 (2021) 102218

Table 1
Chemical composition of the extracted collagen from Sharpnose stingray skin (Dasyatis zugei).

Chemical composition (%)

Samples
Moisture Protein Fat Ash

UASC 4.04 ± 0.10a 41.77 ± 0.47a 4.16 ± 0.10a 46.12 ± 0.25a

UPSC 3.87 ± 0.06a 36.64 ± 0.08b 3.96 ± 0.09a 49.25 ± 0.25a

Sample's collagen defines UASC: Acid Soluble Collagen with ultrasound treatment and UPSC: Pepsin Soluble Collagen with ultrasound treatment. The data represents
(mean ± standard deviation). Different superscript letter(a-b) shows significant difference (p < 0.05) within column.

banded bamboo shark were 89.81 ± 1.91% and 89.89 ± 1.70%, respectively. Similarly, the protein content was lower than the col-
lagen from sharpnose stingray skin, in which protein content for ASC (66.62 ± 2.06%) and PSC (50.96 ± 2.19%) when compared to
the current study (Ong et al., 2021).
The fat content of the extracted collagen did not show any significant difference (p > 0.05) between the UASC (4.16 ± 0.10%)
and the UPSC (3.96 ± 0.09%) from Sharpnose stingray skin. However, the fat content of UASC and UPSC higher than the without ul-
trasound-treated ASC (0.57 ± 0.01%) and PSC (0.25 ± 0.02%). Moreover, these findings were higher compared to previous study
by Wang et al. (2013), who found that the fat content of ASC and PSC from scales of croceine croaker was 0.43 ± 0.15% and
0.15 ± 0.08%, respectively. A similar study was reported by HukmiSarbon (2018) that, the fat content of ASC and PSC from silver
catfish skin was 0.81 ± 0.32% and 0.92 ± 0.38%, respectively. The differences with previous findings may be due to the use of dif-
ferent type and parts of fish, different environmental quality of its habitat as well as the absence of the pre-treatment of defatting
process (Porto et al., 2016).
Similarly, there was no significant differences (p > 0.05) between the ash content of UASC and UPSC from Sharpnose stingray
skin. The ash content of UASC was 46.12 ± 0.25%, while the ash content of UPSC was 49.25 ± 0.25% which is significantly higher
than the ASC (27.00 ± 0.83%) and PSC (30 0.00 ± 1.9%) without ultrasonic-treatment. The high ash content of UASC and UPSC
could be due to the lack of desalting treatment after salting out by NaCl. The purpose of demineralization process, is to decrease the
content of mineral in the raw skin. This had a significant impact on the extraction process. Moreover, the higher ash content in the
skin was associated with the skin thickness level and its biochemical composition (Muralidharan et al., 2013). However, according
Wang et al. (2013) study, the ash content of ASC and PSC from scales of redlip croaker was 1.09 ± 0.05% and 1.13 ± 0.05%, respec-
tively.

3.3. Physical properties

3.3.1. Electrophoretic pattern
The electrophoretic pattern of extracted UASC and UPSC from Sharpnose stingray skin and their respective molecular weight de-
picted in Fig. 1. The electrophoretic pattern shown by both UASC and UPSC extracts were not influence by extraction method.
The molecular weight of α-2 chains in the extracted UASC and UPSC was 100 kDa and 95 kDa, respectively. The lower molecular
weight of α-2 chains in UPSC was due to pepsin action on skin of Sharpnose stingray increasing the degree of hydrolysis, leading to
more destructive effects onto the hydrogen bond in UPSC helix structure (Kittiphattanabawon et al., 2010). It's also obvious that en-

Fig. 1. SDS-PAGE pattern of UASC (UASC 1 and 2) and UPSC (UPSC 1 and 2) extracted from the skin of Sharpnose stingray and marker protein.

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M.I. Shaik et al. Biocatalysis and Agricultural Biotechnology 38 (2021) 102218

zymes can break down protein macromolecules. In terms of band intensity, the higher the intra- and inter-cross-linking within the
collagen structure, the higher the band intensity (Krishnamoorthi et al., 2017). UPSC exhibited a lower content of cross-linking in the
collagen structure due to the activity of pepsin in hydrolyzing telopeptide region and contributing to the destruction of hydrogen
bonding between triple helix structures of collagen (Kittiphattanabawon et al., 2010). β-chain and γ-chain were missing in the both
UASC and UPSC extracts. According to Kittiphattanabawon et al. (2010), the presence of β-chain and γ-chain along the α-chain indi-
cates the absence of disulphite bonds in the extracted collagen. Therefore, the absence of both β-chain and γ-chain indicates the pres-
ence of disulphite bonds in the UASC and UPSC extracts. The findings of the current study were similar to the study conducted by
Krishnamoorthi et al. (2017) on extracted ASC from the skin of sepia pharaonis with molecular weight of 97 kDa. These findings are
supported with the findings of Singh et al. (2011) for the α-2 chain from skin of striped catfish. The molecular weight of the α-2 chain
for ASC (118 kDa) was slightly higher than that of the PSC (116 kDa).

3.3.2. Morphological properties

The microstructure of extracted UASC and UPSC from Sharpnose stingray skin presented in Fig. 2. Both showed a similar struc-
ture, forming a loose, irregular dense sheet-like film, and a less uniform, scaffold-like structure with irregular filamentous pattern and
irregular pore size. The irregular pore formation was caused by the evaporation of fluids from the collagen surface and this process is
independent of the extraction method (Li, 2014).
Findings by Ong et al. (2021) for ASC and PSC from Sharpnose stingray skin with a surface morphology showed a dense, uneven
and multilayer aggregated structure. In addition, the pore sizes of the ASC and PSC were less orderly and smaller compared to those of
UASC and UPSC. In general, the micrograph of ASC appeared to be spongy, small and irregularly distributed whereas UASC showed
porous patterns with a meshwork appearance and regular-shaped caves due to the cavitation and mechanical oscillations of the ultra-
sound wave (Zou et al., 2017). The surface morphologies of extracted UASC and UPSC from Sharpnose stingray skin were in agree-
ment with the surface morphology of the extracted ASC and PSC by HukmiSarbon (2018), as both the ASC and PSC showed a flaky
and porous structure.

3.3.3. Structural properties

The structural properties of the UASC and UPSC from the Sharpnose stingray skin by using the Fourier transform infrared (FTIR)
showed in Table 2. The FTIR spectra of both extracted UASC and UPSC exhibited the characteristics peaks of Amide A, B, I, II and III
(Fig. 3). There were no significant differences between the extracted UASC and UPSC in terms of wavenumber and mode of vibration.

Fig. 2. Surface morphology of (A1) UASC at magnification of 1000 × (A2) UASC at magnification of 400 × (B1) UPSC at magnification of 1000 × and (B2) UPSC at
magnification of 400 × extracted from skin of Sharpnose stingray.

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Table 2
Major functional group found in UASC and UPSC of Shapnose stingray skin.

Functional group Mode of vibration Wavelength (cm−1)

UASC UPSC

Amide A N–H stretching 3443.19 ± 0.22a 3449.05 ± 6.29a

Amide B C–H stretching 2996.98 ± 3.01a 2998.11 ± 5.42a
Amide I C=O stretching 1632.36 ± 3.61a 1636.63 ± 0.15a
Amide II N–H bending 1573.36 ± 5.43a 1578.63 ± 0.63a
Amide III N–H bending 1262.96 ± 0.08a 1261.75 ± 0.76a

Sample's collagen defines UASC: Acid Soluble Collagen with ultrasound treatment and UPSC: Pepsin Soluble Collagen with ultrasound treatment. The data represents
(mean ± standard deviation). Similar superscript letter shows no significant difference (p > 0.05) within column.

Fig. 3. Fourier transform infrared spectra of native collagen derived as ultrasound assisted extraction acid soluble collagen (UASC) and ultrasound assisted extraction
pepsin soluble collagen (UPSC) derived from Sharpnose stingray skin.

The amide A showed no significant differences in the wavenumber between the extracted UASC and UPSC, which is associated
with N–H stretching vibration (Table 2). The presence of amide A functional group indicated the presence of N–H group to be in-
volved in hydrogen bonds for protein structure in both UASC and UPSC (Matmaroh et al., 2011). These findings were supported with
a study conducted by Ong et al. (2021), who found that the amide A bands of Sharpnose stingray skin for ASC and PSC were observed
at 3423.55 cm−1 and 3439.12 cm−1, respectively, with N–H stretching. Similar findings were obtained for the structural properties of
ASC, UASC, PSC and UPSC in terms of wavenumber and mode of vibration. This is in line with the study carried out by Ali et al.
(2018), on the skin of golden carp (Probarbus jullieni) in which the wavenumber for ASC, UASC, PSC and UPSC were 3301 cm−1,
3300 cm−1, 3296 cm−1 and 3295 cm−1, respectively.
Moreover, amide B associated with asymmetrical stretching of CH2 of UASC and UPSC (Table 2). The current finding was sup-
ported by a study on clown featherback (Chitala ornata) skin in which the wavenumber of 2926 cm−1 and 2922 cm−1 for UAP-80/10-
C UAP-80/30-C, respectively (Petcharat et al., 2020). Similarly, amide B peaks in the ASC and UASC form skin of golden carp were
observed at 2923 and 2923 cm−1, respectively, with asymmetrical stretching of CH2 (Ali et al., 2018).
In addition, there were no significant differences between the extracted UASC and UPSC observed for amide I which associated
with C=O stretching vibration (Table 2). The frequencies of amide I are proportional to the degree of molecular order (Muyonga et
al., 2004). Therefore, this indicated that, the molecular order in both UASC and UPSC were similar. According to Ong et al. (2021),
amide I peaks in Sharpnose stingray skin for ASC and PSC were observed at 1626.81 cm−1 and 1627.17 cm−1, respectively. This
showed that the collagen extracted from stingray skin with or without the use of ultrasound treatment had a similar wavelength.
These findings were matched with the study conducted by Ali et al. (2018), in which, the amide I peaks from golden carp skin for
ASC, UASC, PSC and UPSC were 1639 cm−1, 1636 cm−1, 1631 cm−1, 1630 cm−1, respectively.
Similarly, the amide II also showed no significant differences between extracted UASC and UPSC which associated with N–H
bending vibration (Table 2). The wavenumber of amide II was related to the strength of collagen structure (Sanden, 2011). This can
be explained by the similar strength of the collagen structure between UASC and UPSC. However, findings by Ong et al. (2021)
showed that, the amide II for ASC and PSC from Sharpnose stingray skin were 1552.93 cm−1 and 1543.90 cm−1, respectively. The
higher wavenumber obtained (Table 2) compared to the Ong et al. (2021) study, indicates a higher strength of collagen structure for
UASC and UPSC. These findings were corresponded to the thermal stability of ASC, UASC, PSC and UPSC in terms of maximum tem-
perature (Tmax).
Furthermore, amide III showed no significant difference between the extracted UASC and UPSC, as it was associated with N–H
bending vibration (Table 2). The presence of amide III band was an indicator for the presence of the triple helix structure in collagen.
The amide III obtained for UASC and UPSC in this study did not significantly differ from the results obtained by Ong et al. (2021) on
ASC (1239.33 cm−1) and PSC (1238.81 cm−1) of the stingray skin. These findings were similar to the findings obtained by Ali et al.

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M.I. Shaik et al. Biocatalysis and Agricultural Biotechnology 38 (2021) 102218

(2018) for the golden carp skin which the amide III peaks for ASC, UASC, PSC and UPSC were 1236 cm−1, 1236 cm−1, 1236 cm−1 and
1236 cm−1, respectively. These findings were indicated that the presence of a triple helix structure was not affected by the ultrasound
treatment. These results are consistent with the findings of the morphological analysis (Fig. 2), which found no difference between
the UASC and UPSC in terms of their surface morphology.

3.3.4. Thermal stability

The thermal stability of UASC and UPSC extracted from the Sharpnose stingray skins were demonstrated in terms of denaturation
temperature (Td) and maximum temperature (Tmax). The maximum temperature values from differential scanning calorimeter (DSC)
showed in Table 3. There were no significant differences between the maximum temperatures for the UASC (45.57 ± 0.00) and the
UPSC (45.55 ± 1.40). The similar maximum temperature between UASC and UPSC indicated that pepsin extraction did not affect
collagen structure, and especially the triple helical structure. Maximum temperature differences were primarily due to imino acid
content, body temperature, and temperature of the fish environment (Nagai and Suzuki, 2008). Therefore, the same species of fish be-
ing used for extraction of UASC and UPSC meant that the maximum temperatures did not differ. These findings were supported by
Kittiphattanabawon et al. (2010) on the collagen extracted from skin of brownbanded bamboo shark, for which the maximum tem-
perature for ASC and PSC was 34.45 °C and 34.52 °C, respectively. The application of ultrasound treatment did not influence the max-
imum temperatures of the UASC and UPSC.
According to Ong et al. (2021), the maximum temperatures for ASC and PSC from Sharpnose stingray skins were 31.94 ± 0.13 °C
and 31.79 ± 0.23 °C, respectively. The current findings showed a higher maximum temperature as compared to findings by Ong et
al. (2021). The higher maximum temperature for the extracted collagen with ultrasound treatment was due to the ultrasonic cavita-
tion effects by breaking the cell envelope of the particle substance. For UPSC, this permitted more of the pepsin to pass through to
cleave the telopeptide region effectively. These findings are validated by the study conducted by Ali et al. (2018), in which the maxi-
mum temperatures for PSC and UPSC from the skins of golden carp were 38.27 °C and 40.82 °C, respectively.

3.3.5. Viscosity
The relative viscosity of extracted UASC and UPSC from Sharpnose stingray skin within a temperature range of 4–48 °C shown in
Fig. 4. The relative viscosity of the UASC and UPSC decreased as the temperature increased. However, there was a slightly difference
between the UASC and UPSC with respect to relative viscosity trends. At temperature ranges of between 4 and 16 °C, the relative vis-
cosity of UASC decreased slightly and had a drastically decreased when the temperature was increased from 16 to 44 °C. The UASC
remained low viscosity after being heated to above 44 °C. For UPSC, the relative viscosity of extracted collagen from Sharpnose

Table 3
Maximum temperature (Tmax) and enthalpy of UASC and UPSC from sharpnose stingray skin.

Collagen Tmax (°C)

UASC 45.57 ± 0.00a

UPSC 45.55 ± 1.40a

Samples collagen defines UASC: Acid Soluble Collagen with ultrasound treatment and UPSC: Pepsin Soluble Collagen with ultrasound treatment. The data represents
(mean ± standard deviation). Similar superscript letter shows no significant difference (p > 0.05) within column.

Fig. 4. Relative viscosity of UASC and UPSC of Sharpnose stingray skin.

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M.I. Shaik et al. Biocatalysis and Agricultural Biotechnology 38 (2021) 102218

Fig. 5. Solubility of extracted UASC and UPSC of Sharpnose stingray skin in pH range of 1–10.

stingray skin decreased gradually from 4 to 36 °C, and it experienced a rapid decrease in relative viscosity when temperatures were
above 36 °C. The thermal denaturation temperature (Td) of ASC and PSC from eel fish skin collagen showed transition curves at
38.5 °C and 35.0 °C, respectively, which is lower than that of current study (Veeruraj et al., 2013). This study suggested that intramol-
ecular hydrogen bonds stabilizing the triple helix structure of collagen could be disrupted to some levels in presence of acetic acid.
Higher temperatures reduce the viscosity of the solution, thereby increasing the mass transfer rate. In addition, the increase in tem-
perature has improved the purity of collagen until a minimum point of 55 °C was reached (Jafari et al., 2020).
The relative viscosity of the extracted collagen decreased when it was heated as heat energy gradually broke the hydrogen bonds
of collagen molecule and led to the conversion of triple helix structure into a random coil configuration through a process of thermal
depolymerisation (Ong et al., 2021). At temperatures above 44 °C, the relative viscosity of both extracted collagens remained con-
stant due to the complete uncoiling of the collagen protein structure. Collagen protein molecules were completely denatured when
collagen was heated to a temperature greater than the denaturing temperature (Duan et al., 2009). The breaking of the collagen struc-
ture will lead to physical changes, such as viscosity, sedimentation, diffusion, light scattering, and so on (Wang et al., 2008).
The relative viscosity trends of both extracted UASC and UPSC from Sharpnose stingray skin were in agreement with a study by
Huang et al. (2011). According to Huang et al. (2011), there was no significant difference between the viscosity trends of ASC and
PSC from balloon fish skin. There was a gradual decrease in relative viscosity when it was heated from 15 °C to 40 °C. Temperatures
above 40 °C allowed the observation of constant relative viscosity. This indicated that at above denaturation temperature, the inter-
molecular bonds in both UASC and UPSC were broken down completely and therefore led to a constant viscosity regardless of further
increase in temperature (Kaewdang et al., 2014). This finding indicates that the extraction method did not affected trends in relative
viscosity trends of the extracted collagen from Sharpnose stingray skin.

3.3.6. Solubility
The relative solubility levels of the UASC and USPC from the skin of Sharpnose stingray in the pH range of 1–10 illustrated in Fig.
5. Both extracted UASC and UPSC exhibited higher solubility under acidic conditions (a pH range of 1–5). The lowest solubility of ex-
tracted UASC and UPSC was observed at pH 8, followed by slightly increase in the solubility at basic pH. In general, there was no dif-
ferences between UASC and UPSC from skin of Sharpnose stingray in terms of their trends of solubility at different pH.
The extracted collagen solubility was higher in acidic condition and with minimum solubility at pH 8, followed by increased solu-
bility under base conditions. The lowest solubility at pH 8 was caused by the hydrophobic interaction between collagen molecules. In
other words, both UASC and UPSC had their isoelectric point at pH 8. At the iso-electric point, hydrophobic-hydrophobic interaction
increased and contributed to collagen aggregation and precipitation. As a result, it led to a decline in solubility (Chi et al., 2013). In
addition, the increase in the solubility in base condition was due to the repulsion forces between polypeptide chains as the pH was
higher than isoelectric points (Nalinanon et al., 2007). UPSC had higher solubility compared to UASC at almost all pH levels, except
for pH 6 and 10, due to the lower degree of cross-linking and a weaker bond in UPSC compared to UASC. Pepsin extraction could re-
duce the degree of crosslinking or weaken bonds compared to UASC. In other words, UASC contained higher molecular weight cross-
linkages that cannot be broken down with acetic acid. This may be correlated with the findings from the molecular weight-based elec-
trophoretic model (Fig. 1).
The solubility trends were in agreement with findings by Jeevithan et al. (2014) on the skeletons and head bones of sharks, for
which both extracted ASC and PSC had the highest solubility at pH 5 and decreased solubility at pH 8 and 9. Similar findings were
also shown by Chi et al. (2013) for the cartilage of red stingray, at which the extracted ASC had the highest solubility at pH 2 and re-

9
M.I. Shaik et al. Biocatalysis and Agricultural Biotechnology 38 (2021) 102218

mained soluble at pH 1 to 4, whereas the minimum solubility was observed at pH 7, followed by a further increase in solubility with
increasing alkalinity.

4. Conclusion
Compared to other marine species including fishes, corals and sponges, the yield of Stingray species is high and application of ul-
trasound technique enhanced the collagen yield. The ultrasound-assisted extraction method showed a better extractability of pepsin
soluble collagen (UPSC) and acid soluble collagen (UASC) in shorter time. However, there was no significant difference in chemical
composition such as moisture, fat and ash for both UPSC and UASC except for the protein content. The enzymatic extraction of colla-
gen by pepsin resulted in low molecular weight electrophoretic pattern obtained by UPSC with the presence of α-2 chain. The current
results demonstrated that, there were no significant differences in morphology, structural properties, thermal stability, relative vis-
cosity and solubility for both UASC and UPSC. The novelty of the current study is that, the ultrasound-assisted extraction method has
improved the extractability of Sharpnose stingray skin collagen without affecting the properties. In conclusion, the application of the
ultrasound-assisted extraction method suggested to obtain a higher yield of collagen extraction with better physicochemical proper-
ties.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to
influence the work reported in this paper.

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