LWT - Food Science and Technology 139 (2021) 110502

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

Proteolysis of tilapia skin collagen: Identification and release behavior of

ACE-inhibitory peptides
Junde Chen a, *, Shanshan Sun a, Yushuang Li a, Rui Liu b
a
Technology Innovation Center for Exploitation of Marine Biological Resources, Third Institute of Oceanography, Ministry of Natural Resources, Xiamen, 361005, China
b
Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, And National and Local Collaborative Engineering Center of Chinese
Medicinal Resources Industrialization and Formulae Innovative Medicine, Nanjing University of Chinese Medicine, Nanjing, 210023, China

A R T I C L E I N F O A B S T R A C T

Keywords: The structure–activity relationship (SAR) and release behavior of angiotensin I-converting enzyme inhibitory
Collagen (ACEi) peptides obtained from enzymolysis of collagen influence the large-scale production of ACEi peptides.
ACE inhibitory Peptides However, researchers have paid insufficient attention to these areas. In this study, we extracted collagen from
Release behavior
tilapia skin, and hydrolyzed it using three proteases. A total of 270 peptides were released from the collagen
Structure–activity relationship
parent protein. The SAR of these larger collagen ACEi peptides indicated that the presence of proline at position
C2 of three C-terminal sequences has a greater effect on increasing the ACEi activity of the peptide than at
position C1. The release behavior of these collagen peptides showed that bromelain and alcalase preferentially
cleave the N-terminal region of the collagen α1 subunit and then the C-terminal region. These enzymes evenly
cleave regions of the collagen α2 subunit. Collagenase preferentially cleaves the C-terminal region of the collagen
subunit, followed by the N-terminal region, and then the middle region. The pattern of peptide release from
different proteases and the SAR of larger collagen peptides can help guide food production processes to ensure
food safety, and to produce high-quality active peptide products.

1. Introduction
Collagen is the most abundant fibrous protein in animals and ac­
counts for about 30% of the total protein content (Abdollahi, Rezaei,
Jafarpour, & Undeland, 2018). Mammalian collagen is potentially
associated with infectious diseases such as avian and swine influenza,
bovine spongiform encephalophy, and foot-and-mouth disease, which
can cross-infect terrestrial animals and humans (Shi et al., 2019).
Mammalian collagen has much higher crosslinking and advanced gly­
cation end products than marine collagen. This causes low yields that
increases the cost of collagen production from terrestrial vertebrates
compared to marine species (Hong, Fan, Chalamaiah, & Wu, 2019).
Therefore, collagen derived from fish byproducts (mainly fish scales and
fish skin) is an attractive alternative collagen source. There is great
demand for fish by-product-derived collagen, and this is now the main
source of collagen globally (Chen, Li, Yi, Gao, & He, 2018).
The molecular structure of fish collagen is a three-helical structure
formed by three collagen subunits through right-handed coils. Each
collagen subunit is composed of more than 1000 amino acids (Sun, Hou,
Li, & Zhang, 2017). Bioactive peptides appear as inactive sequences in
the collagen subunit parent protein and are released by hydrolysis using
proteases (He, Franco, & Zhang, 2013). Food scientists have used
commercial proteases to hydrolyze the fish collagen parent proteins to
obtain collagen peptides. These peptides have wound -healing, antiox­
idant, and antifreeze activities, and have been used in health and
nutritional supplement products (Sato, 2017; Wu et al., 2018). In 2019,
the total market value of collagen peptides exceeded 100 million US
dollars and is expected to grow even larger (Hong et al. 2019).
Collagen peptides have greater stability during gastrointestinal
digestion and intestinal transport due to the presence of proline and the
peptides with a proline residue at the C-terminus are good candidates for
potent ACE inhibitors. Collagen angiotensin I-converting enzyme
inhibitory (ACEi) peptide is a promising collagen active peptide in the
market. ACEi peptides can prevent and treat hypertension by blocking
the activation of the renin-angiotensin system (Udenigwe & Mohan,
2014). However, compared with other high value-added functional
peptides, research and development of collagen ACEi peptides in in­
dustrial production is slow, and collagen ACEi peptides are rarely seen in
the market. It is essential to understand the release mechanism of
collagen ACEi peptides. This can guide the production of high-quality

* Corresponding author.
E-mail addresses: jdchen@tio.org.cn (J. Chen), shshsun123@163.com (S. Sun), wwwwaway@163.com (Y. Li), cpulr@126.com (R. Liu).

https://doi.org/10.1016/j.lwt.2020.110502
Received 15 June 2020; Received in revised form 15 October 2020; Accepted 31 October 2020
Available online 2 November 2020
0023-6438/© 2020 Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
J. Chen et al.
collagen ACEi peptides. However, the reaction behavior of collagen
chains during enzymatic hydrolysis has not been studied. The amino
acid sequence of collagen peptides and the structure–activity relation­
ship (SAR) between collagen peptides and ACEi activity are also un­
known, preventing the quality improvement of collagen ACEi peptides.
The SAR of ACEi short peptides (di- and tripeptides) has been exten­
sively studied. The SAR of synthetic ACEi short peptides indicates that
amino acid residues at the three positions of the peptide C-terminal site
greatly influence peptide ACEi activity (Li, Le, Shi, & Shrestha, 2004).
The amino acid residues with bulky and hydrophobic side chains are key
to determining the peptide ACEi activity of dipeptides (Wu, Aluko, &
Nakai, 2006). Tripeptides with ACEi activity have aromatic amino acids
at their C-terminal, positively charged amino acids at their middle po­
sition, and hydrophobic amino acids at the N-terminal (Fan, Liao, & Wu,
2019; Wu et al., 2006). The SAR of larger ACEi peptides (>3 amino
acids) have been studied. Fan, Wang, Liao, Jiang, and Wu (2019) studied
egg white peptides obtained from enzymolysis. The potent penta- and
hexapeptides preferentially harbor both a hydrophobic N-terminus and
a hydrophobic tetrapeptide C-terminus, and the positive charges at the
second and fourth positions from the C-terminus enhance ACEi activity.
Other studies have demonstrated that the C-terminal tripeptide of most
ACEi peptides (short peptides and larger peptides) contain proline
(Abdelhedi et al., 2018). However, there are no studies on the SAR of
larger ACEi peptides derived via enzymolysis of collagen. Proteomics
and peptidomics, liquid chromatography-tandem mass spectrometry
(LC-MS/MS) analysis and related database searches, are useful methods
for analyzing proteins and peptides in samples with complex composi­
tions. These methods allow for the identification of proteins and pep­
tides in mixtures, and can be used to identify collagen and ACEi peptides
obtained from the enzymolysis of collagen (Li et al., 2017; Liu et al.,
2017; Song & Mechref, 2013).
In this study we extracted tilapia skin collagen and conducted pro­
teomics analysis to determine its amino acid sequence. The collagen
parent protein was then enzymolyzed with commercial proteases. For
the first time, peptidomics was used to identify tilapia skin collagen
ACEi peptides. Then, the SAR and release behavior of collagen ACEi
peptides were analyzed. The study goal was to determine the release
behavior of ACEi peptides, analyze the SAR of collagen peptides, and
then develop possible methods for large-scale production of collagen
ACEi peptides.
2. Materials and methods
2.1. Materials
Tilapia (Oreochromis mossambicus) skin was purchased from an
aquatic product processing plant in Zhangzhou, China. High-molecular
weight markers, ACE, hippuryl-L-histidyl-L-leucine (HHL), acetonitrile,
sequencing grade trypsin, collagenase, formic acid, and trifluoroacetic
acid (TFA) were obtained from Sigma Chemical Co. (St. Louis, MO,
USA). Coomassie blue R-250 was obtained from Bio-Rad Laboratories
(Hercules, CA, USA). Bromelain (2 × 106 U/g), collagenase (2 × 106 U/
g), alcalase (2 × 106 U/g), trypsin (2 × 106 U/g), neutrase (2 × 106 U/g),
papain (2 × 106 U/g), acid protease (2 × 106 U/g), and pepsin (2 × 106
U/g) were purchased from Nanning Pangbo Biological Engineering Co.,
Ltd. (Nanning, China). All other chemicals and reagents used in this
study were of analytical grade.
2.2. Preparation of collagen parent protein
Collagen was prepared as described by Cao, Duan, Liu, Shen, and Li
(2019), with slight modifications. Tilapia skin was immersed in 0.1%
(w/v) sodium bicarbonate at a ratio of 1:10 (w/v), and the mixture was
stirred for 2 h to remove non-collagen components. Subsequently, 0.5
mol/L acetic acid containing 1% pepsin (w/w) was added to the skin at a
ratio of 1:10 (w/v), and the mixture was stirred for 24 h. Then, the
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LWT 139 (2021) 110502
collagen extract was centrifuged at 20,000 g for 15 min. The salting-out
of the collagen extract supernatant was conducted by adding 3% (w/v)
NaCl, and the precipitate was collected after centrifugation at 10,000 g
for 20 min. The precipitate was dissolved in 0.5 mol/L acetic acid at a
ratio of 1:10 (w/v), and then dialyzed in 20 vol of 0.1 mol/L acetic acid
for 24 h, followed by dialysis in distilled water for 24 h. All processes
were performed below 4 ◦ C. Finally, the collagen was freeze-dried in a
freeze dryer (Lyobeta 25, Telstar, Spain) and stored at − 20 ◦ C until
analysis.
2.3. Structural characterization of collagen parent proteins
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) was conducted according to Chen et al. (2019). A mini-protein
vertical slab electrophoresis system (Bio-Rad Laboratories, CA, USA)
was used. High-molecular weight markers were used to evaluate the
molecular weight of each sample. The staining ratio of α1 to α2 was
determined using Quantity One 4.6.0 (Bio-Rad Laboratories, CA, USA).
2.4. Collagen proteolysis
Collagen was dissolved in 0.1 mol/L phosphate buffer solution.
Bromelain (50 ◦ C, pH 7.0), alcalase (55 ◦ C, pH 8.0), collagenase (37 ◦ C,
pH7.5), trypsin (37 ◦ C, pH 8.0), neutrase (50 ◦ C, pH 7.0), papain (55 ◦ C,
pH 6.0), acid protease (40 ◦ C, pH 3.0), and pepsin (40 ◦ C, pH 2.0) were
used in the enzymatic hydrolysis of collagen, which was performed for 4
h. The collagen/solution ratio was 4:100 (w/w), and the E/S ratio was
1:100 (w/w). The enzymatic hydrolysate was centrifuged for 20 min
(20,000 g). The supernatant was then freeze-dried in a freeze dryer
(Lyobeta 25, Telstar, Spain) to obtain the collagen peptides.
2.5. Collagen peptide identification by nano-LC-MS/MS
Collagen peptide identification was performed according to Liu et al.
(2017). All MS/MS spectral data were processed using Xcalibur software
(version 2.0.7.), and collagen peptides were identified using PEAKS 8.5
(Bioinformatics Solutions Inc., Waterloo, ON, Canada). The de novo
sequence was used to identify the peptide sequence to construct the data
set, and its average local confidence (ALC) score was >90%. The iden­
tified peptides were filtered to create a data set with a − 10 log P value >
60.
2.6. Measurement of ACE inhibition
ACEi activity was measured according to the method reported by
Liao et al. (2018) with modifications. Collagen peptide samples and
Hip-His-Leu were dissolved in 100 mmol/L boric acid buffer (pH 8.3).
Subsequently, 25 μL of the collagen peptide sample and 75 μL of 60
min/mL ACE solution were used, followed by the addition of 225 μL of
2.5 mmol/L Hip-His-Leu. The mixture was incubated in a 37 ◦ C water
bath for 60 min, and then 25 μL of 0.1% TFA was added to stop the
reaction. Reverse phase-HPLC (RP-HPLC) was used to measure the
content of hippuric acid (HA) in the reaction product. The liquid chro­
matography column was a CAPCELL PAK C18 MG column (4.6 × 250
mm ID, Shiiseido Co., Ltd., Tokyo, Japan). The mobile phase was 25%
acetonitrile solution containing 0.1% TFA, the flow rate was 1 mL/min,
and the UV absorption wavelength was 228 nm. The inhibitory activity
of ACE was calculated using the following equation:
IP = [(A0 − A)/A0] × 100%
where IP is the ACE inhibition percentage of the sample (%); and A and
A0 are chromatographic peak areas of HA with and without sample,
respectively. IC50 values were defined as the concentration of the sample
required to inhibit 50% of the ACE activity.
J. Chen et al.
Fig. 1. SDS-PAGE profiles of skin collagen. Line 1: marker, Line 2: collagen.
3. Results and discussions
3.1. Structural identification of collagen parent protein
SDS-PAGE data of O. niloticus skin collagen are shown in Fig. 1. The
skin collagen contained two different α chains (α1 and α2), and the gray
level ratio of α1 to α2 was close to 2. These electrophoresis images were
similar to the type I collagen of other fish (Chen et al., 2019; Liu, Liang,
Regenstein, & Zhou, 2012; Wang, Pei, Liu, & Zhou, 2018). These results
indicated that O. niloticus skin collagen should be classified as a type I
collagen that contains, two identical α1 chains and one α2 chain in the
molecular form of [α1(I)]2α2(I). Skin collagen also contained dimer β
chains and trimer ϒ chains formed by intramolecular and intermolecular
cross-links of collagen. Quantification of stained protein bands showed
that, α2, β and γ subunits accounted for 98% of the collagen sample.
These data indicated that pepsin does not destroy the triple helical
structure of collagen during collagen extraction. Additionally, according
to our previous study (Chen, Yi, Xu, Gao, & Hong, 2016), the protein
sequence of O. niloticus skin collagen subunit is shown in Supplementary
Materials 1 and 2.
3.2. Characterization of collagen ACEi peptides
3.2.1. SAR analysis of ACEi peptides
Collagen was hydrolyzed using eight proteases, and ACEi activity
was evaluated using IC50 values. The alcalase enzymolysis peptides
(AEPs) exhibited the highest ACEi activity (IC50 = 0.14 ± 0.03 mg/mL),
followed by collagenase enzymolysis peptides (CEPs) (IC50 = 0.19 ±
0.05 mg/mL), and bromelain enzymolysis peptides (BEPs) (IC50 = 0.27
± 0.02 mg/mL). Trypsin, neutrase, papain, acid protease, and pepsin
enzymolysis peptides showed weak ACEi activity, with IC50 values of
5.21 ± 0.07, 6.72 ± 0.05, 12.62 ± 0.03, 14.92 ± 0.12, and 15.62 ± 0.08
mg/mL, respectively. The ACEi activity of AEPs, CEPs, and BEPs were
higher than the hydrolysates of bovine caseins (IC50 = 1.72 ± 0.25 mg/
mL), hydrolysates of milk protein (IC50 = 0.74 mg/mL), and protein
hydrolysates of shrimp waste (IC50 = 2.17 mg/mL) (Corrons, Liggieri,
Trejo, & Bruno, 2017; Huang et al., 2020; Krichen et al., 2018). Thus,
AEPs, CEPs, and BEPs were selected for further study.
After enzymolysis of the collagen parent protein by bromelain, a
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LWT 139 (2021) 110502
Table 1
Proportion of peptides with proline at the three C-terminal positions of collagen
chains.
Collagen enzymolysis peptides C1a (%) C2b (%) C3c (%)
BEPs 50 12.2 0
CEPs 6.92 21.54 4.62
AEPs 0 57.38 4.92
a
C1 refers to the position of the third last amino acid residue at the three C-
terminus of collagen subunit.
b
C2 refers to the position of the second last amino acid residue at the three C-
terminus of the collagen subunit.
c
C3 refers to the position of the last amino acid residue at the three C-terminus
of the collagen subunit.
total of 82 peptides were released, including 60 peptides derived from
collagen α1 subunit and 22 peptides derived from collagen α2 subunit
(Supplementary materials 3, 4). After enzymolysis of the collagen parent
protein by alcalase, a total of 58 peptides were released, including 38
peptides derived from collagen α1 subunit and 20 peptides derived from
collagen α2 subunit (Supplementary materials 5, 6). After enzymolysis of
the collagen parent protein by collagenase, a total of 130 peptides were
released, including 66 peptides derived from collagen α1 subunit and 64
peptides derived from collagen α2 subunit (Supplementary materials 7,
8). All peptides obtained from enzymolysis of tilapia skin collagen are
large collagen peptides (>3 amino acids). Tilapia skin collagen is
composed of two α1 chains and one α2 chain, and each polypeptide chain
contains the repeated typical tripeptide units Gly-X-Y, where Gly is
glycine, and X and Y are often proline and hydroxyproline, respectively
(Supplementary materials 1, 2). The conservative sequence (Gly-X-Y)n
may protect collagen from being digested by proteases into di- and tri­
peptides. Additionally, the proline may be another important factor that
protects collagen from being digested by protease into di- and
tripeptides.
To assess the ACEi activity of the released peptides, we statistically
analyzed the composition of the last three amino acids at the C-terminal
of the 270 released peptides. The amino acid residues at the three C-
terminal positons of each peptide were counted. In Table 1, C1 refers to
the position of the third to the last amino acid residue at the C-terminus
of the collagen subunits; C2 refers to the position of the second to the last
amino acid residue at the C-terminus of the collagen subunits; and C3
refers to the position of the last amino acid residue at the C-terminus of
the collagen subunits. Among the three collagen enzymolysis peptides,
BEPs had the highest proportion of peptides with proline at position C1
(50%), followed by CEPs (6.92%), and AEPs (0%) (Table 1). AEPs had
the highest proportion of peptides with proline at position C2 (57.38%),
followed by CEPs (21.54%), and BEPs (12.21%). AEPs showed the
highest ACEi activity, followed by CEPs, and BEPs. These data indicated
that in the collagen peptides obtained from enzymolysis of tilapia skin,
proline at position C2 may have a greater effect on the peptide ACEi
activity than that at position C1. Additionally, both BEPs, CEPs and AEPs
had a low proportion of peptides with proline at position C3 (0%, 4.62%,
and 4.92%, respectively).
3.2.2. Prediction of ACEi activity of collagen peptides using AHTpin
platform
To better understand the SAR of tilapia skin collagen peptides, the
web server of AHTpin (http://crdd.osdd.net/raghava/ahtpin/) was used
to predict the ACEi activity of tilapia skin collagen peptides obtained
from enzymolysis. The web server of AHTpin is software developed by
Kumar et al. (2015) to study the ACEi activity of peptides using QSAR
(Kumar et al., 2015). AHTpin was used to analyze the peptides having
anti-hypertensive inhibitory activity by submitting the peptide sequence
to the server. AHTpin predicts the ACEi activity of peptides that contain
more than 12 amino acid residues per molecule. When the peptide ac­
tivity is predicted to be “AHT”, the peptide sequence is considered to
J. Chen et al.
have potential ACEi activity; when the peptide activity is predicted to be
“non-AHT”, the peptide sequence is considered to have no ACEi activity.
The web server can also score peptide activity. The higher the score, the
higher the ACEi activity of the peptide. When the score is > 1, the
peptide may have a high ACEi activity. The prediction results of the
AHTpin platform showed that 46, 23, and 62 peptides, which might
have ACEi activity, were found in BEPs, AEPs, and CEPs, respectively
(Supplementary materials 9–11). There were also 3, 9, and 6 peptides
with SVM scores of >1 in BEPs, AEPs, and CEPs, respectively (Supple­
mentary material 12). The C-terminal tripeptide of all collagen peptides
with a SVM score of >1 contained at least one hydrophobic amino acid.
This indicated that among the tilapia skin collagen peptides, the hy­
drophobic amino acid at the C-terminal tripeptide position of the pep­
tide chain may increase the ACEi activity of the peptide. This is
concordant with the findings of Li et al. (2004) that ACE seems to
preferably select substrates or competitive inhibitors that have
Fig. 3. The hydrophilic and hydrophobic distribution of tilapia skin collagen subunits. A: α1 subunit, B: α2 subunit.
4
LWT 139 (2021) 110502
Fig. 2. Release behaviors of peptides from collagen.
0–1447 refers to the amino acid positions in the
peptide sequence of the α1 subunit; 0–1350 refers to
the amino acid positions in the peptide sequence of
the α2 subunit; A、B and C refer to the release be­
haviors of peptides from collagen α1 subunit by
bromelain、pepsin, and alkaline protease enzymol­
ysis resepctively. C、D and E refer to the release be­
haviors of peptides from collagen α2 subunit by
bromelain、pepsin, and alkaline protease enzymol­
ysis resepctively.
hydrophobic amino acid residues at each of the three C-terminal posi­
tions (Li et al., 2004). Additionally, the C-terminal tripeptide GPM of
tilapia skin collagen peptide EKSPAMPVPGPM (SVM scores 1.13) ob­
tained from enzymolysis was concordant with the ACEi peptide GPM
(IC50 = 17.13 μmol/L) extracted from Alaska cod skin by Byun and Kim
(2001), indicating that the C-terminal tripeptide GPM may increase the
ACEi activity of EKSPAMPVPGPM.
3.3. Release behavior of the ACEi peptides
To explain the release behavior of ACEi peptides, the positions of the
released peptides in the amino acid sequence of collagen parent protein
were counted. Matlab V7.1 software (MathWorks, Natick, MA, USA) was
used for plotting and analysis. The blue area in Fig. 2 indicates that the
peptide chains of collagen parent protein did not undergo enzymatic
hydrolysis. Collagen α1 subunit consisted of 1447 amino acids
J. Chen et al.
(Supplementary Material 1), and collagen α2 subunit comprised 1350
amino acids (Supplementary Material 2). The highlighted regions were
amino acid residues that had undergone enzymatic hydrolysis in
collagen α subunit. A brighter region indicates that the amino acid
residues underwent more frequent enzymatic cleavage.
Supplementary material 3 and Fig. 2 show that when bromelain was
used for enzymolysis of the collagen parent protein, the cleavage sites on
the collagen α1 subunit were concentrated in α1-CN147-178 region at
the N-terminus and α1-CN1156-1199 region at the C-terminus of the
collagen α1 subunit. The Kyte-Doolittle method was used to calculate the
hydrophobicity distribution of collagen α1 subunit (Fig. 3). The hydro­
phobic regions of the collagen α1 subunit were distributed in the C-ter­
minal region and the N-terminal region. This result was consistent with
the enzymatic hydrolysis site. The N-terminal α1-CN147-178 of collagen
α1 subunit released 42 peptides, and the C-terminal α1-CN1156-1199
released 18 peptides. The number of released peptides of N-terminal α1-
CN147-178 was substantially higher than that of C-terminal α1-CN1156-
1199. This demonstrated that bromelain preferentially cleaved the N-
terminal region of the collagen α1 subunit, and then cleaved the C-ter­
minal region. The middle region of the collagen α1 subunit was not
digested by bromelain, indicating that after the bromelain cleaved the C-
terminal and N-terminal regions of collagen α1 subunit, the bromelain
was consumed and it was not possible to further cleave the amino acid
sequence in the middle of α1 subunit. In addition, Supplementary ma­
terial 3 shows that bromelain performed a second enzymolysis of the
peptides obtained by the first enzymolysis of the α1 subunit, thereby
releasing more active peptides. For example, bromelain released
QMSGGYDEKSPAM PVPGPMPRG from the collagen α1 subunit. Further
enzymolysis of this peptide by bromelain released a series of peptides
such as QMSGGYDEKSPAMPVPGPM, GGYDEKSPAM PVPGPM, and
DEKSPAMPVPGPM. Supplementary material 4 and Fig. 2 showed that
the enzyme cleavage sites of bromelain on the collagen α2 subunit were
concentrated in six regions, including α2-CN67-84, α2-CN587-601, α2-
CN713-727, α2-CN812-831, α2-CN896-915, and α2-CN1085-1107. The
enzymatic hydrolytic sites of bromelain in the collagen α2 subunit were
more uniformly distributed in various regions of the α2 subunit. The
Kyte-Doolittle method was used to calculate the hydrophobicity distri­
bution of the collagen α2 subunit (Fig. 3), and the hydrophobic region of
the collagen α2 subunit was distributed at the C-terminus, N-terminus,
and the middle region. This result was consistent with the distribution of
the enzymatic hydrolysis site.
The enzymatic cleavage of collagen by alcalase was similar to that of
bromelain. Alcalase preferentially cleaved the N-terminal region of the
collagen α1 subunit, then cleaved the C-terminal region, and evenly
cleaved each region of the collagen α2 subunit. However, compared with
bromelain, alcalase showed a higher degree of enzymatic hydrolysis,
and it cleaved the α1-CN631-654 region in the middle of the collagen α1
subunit amino acid sequence.
In contrast to the enzymolysis processes of alcalase and bromelain,
collagenase showed the highest degree of enzymolysis to the C-terminal
region of the collagen subunit, followed by the N-terminal region, and
the middle region. Collagenase preferentially cleaved the C-terminal
region of the collagen subunit, followed by the N-terminal region, and
then the middle region. These findings were similar to those of French,
Mookhtiar, and Van Wart (1987), who found that collagenase first
cleaved the C-terminal region of rat tendon collagen and then cleaved
the collagen of N-terminal region of rat tendon collagen.
4. Conclusions
Collagen was extracted from the skin of tilapia and characterized as
type I collagen that contains the [α1(I)]2α2(I) component. Protein se­
quences of collagen sample were identified by nano-LC-MS/MS prote­
omics analyses. Nano-LC-MS/MS peptidomics analyses showed that 82
peptides were released after the enzymolysis of collagen using brome­
lain, 58 peptides were released after the enzymolysis of collagen by
5
LWT 139 (2021) 110502
alcalase, and 130 peptides were released after the enzymolysis of
collagen by collagenase. SAR analysis of these peptides showed that the
presence of proline at position C2 of the collagen peptides might have a
greater effect on increasing ACEi activity of peptides than at position C1.
The prediction of the AHTpin platform indicated that in collagen pep­
tides obtained from enzymolysis of tilapia skin, the presence of hydro­
phobic amino acids at the C-terminal tripeptide position might increase
the ACEi activity of peptides. The release behavior of collagen ACEi
peptides showed that bromelain and alcalase preferentially enzymo­
lyzed the N-terminal region and then the C-terminal region of the
collagen α1 subunit. These enzymes evenly hydrolyzed various regions
of the collagen α2 subunit. In contrast, collagenase preferentially enzy­
molyzed the C-terminal region of the collagen subunit, then the N-ter­
minal region, and finally the middle region. The enzymolysis
mechanisms of these proteases and the SAR of peptides released by
collagen can be used in food production processes to ensure food safety,
and to produce high-quality active peptide products.
CRediT authorship contribution statement
Junde Chen: Formal analysis, Data curation, Data collection, Data
analysis, Software, Writing - original draft, Writing - review & editing,
Funding acquisition. Shanshan Sun: Formal analysis, Data curation,
Data collection, Data analysis, Software. Yushuang Li: Formal analysis,
Data curation, Data analysis, Software. Rui Liu: Formal analysis, Data
curation, Data analysis, Software.
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.
Acknowledgments
This work was supported by grants from the National Natural Science
Foundation of China (41676129, 42076120, 41106149), the Scientific
Research Foundation of the Third Institute of Oceanography (2019010).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.lwt.2020.110502.
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