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Isolation, identification, and molecular docking analysis of novel ACE

inhibitory peptides from Spirulina platensis

Article  in  European Food Research and Technology · April 2022

DOI: 10.1007/s00217-021-03949-x

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European Food Research and Technology (2022) 248:1107–1115
https://doi.org/10.1007/s00217-021-03949-x

ORIGINAL PAPER

Isolation, identification, and molecular docking analysis of novel ACE

inhibitory peptides from Spirulina platensis
Nan Zhang1 · Fuqiang Li1 · Tingxin Zhang1 · Chun‑Yang Li2 · Liping Zhu1 · Shigan Yan1

Received: 17 September 2021 / Revised: 22 December 2021 / Accepted: 26 December 2021 / Published online: 6 January 2022
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022

Abstract
Hypertension has emerged as a serious global health issue and piqued the interest of academics all over the world. Angioten-
sin-I converting enzyme (ACE, EC 3.4.15.1) plays a crucial role in blood pressure regulation. Due to their safety and mildness
compared with synthetic drugs, natural bioactive inhibitory peptides against ACE generated from various protein sources
cause an upsurge of research and development. Spirulina platensis is a cyanobacterium widely distributed in aquaculture and
an ideal protein source for bioactive peptide production, owing to its remarkable protein content and nutritive value. In this
study, novel ACE inhibitory peptides from Spirulina platensis protein hydrolysates digested by commercial thermolysin were
isolated by membrane filtration and size exclusion-high-performance liquid chromatography (SEC-HPLC) in sequence, and
identified by mass spectrum analysis and ACE inhibitory activity assay. The results showed that two novel ACE inhibitory
peptides were obtained, and their amino acid sequences were Val–Thr–Tyr (VTY) and Leu–Gly–Val–Pro (LGVP), with
­IC50 values of 23.39 µM, and 45.76 µM, respectively. Molecular docking analysis indicated that the VTY was mainly bound
in the S1 and S2 active pockets of ACE via hydrogen bonds, while LGVP only partially interacted with the S1 pocket. Our
findings provide novel insights for the development of ACE inhibitors and the high-value utilization of Spirulina platensis.

Keywords  Spirulina platensis · Enzymatic digestion · Angiotensin-I converting enzyme · Inhibitory peptide · Molecular
docking

Introduction and decrease blood pressure. However, these drugs usually

Hypertension has affected over 1.2 billion individuals world-
wide and become a significant public health problem [1].
The renin–angiotensin system (RAS) is important to blood
pressure regulation [2]. In the RAS, angiotensin convert-
ing enzyme (ACE, EC 3.4.15.1) participates in catalyzing
the production of angiotensin II, a potent vasoconstrictor,
and the inactivation of bradykinin, a vasodilator peptide
[3]. Thus, ACE is generally considered as a crucial target
for hypertension treatment. Synthetic chemical drugs (e.g.,
captopril and enalapril), can effectively inhibit ACE activity
* Shigan Yan
yanshigan@126.com
1
State Key Laboratory of Biobased Material and Green
Papermaking, School of Bioengineering, Qilu University
of Technology (Shandong Academy of Sciences),
Jinan 250353, China
2
College of Marine Life Sciences, Ocean University of China,
Qingdao 266100, China
have side effects, such as taste disturbance, skin rashes,
angioedema, and cough [4]. In recent years, natural bio-
active peptides derived from a variety of protein sources,
including animal, plant, and microorganism, have attracted
much attention to serving as potential alternatives or com-
plements to synthetic drugs, owing to their perceived safety
and mildness [5]. A number of ACE inhibitory peptides have
been identified, which commonly contain 2–20 amino acids
[6–14].
Microalgae, such as Spirulina platensis, Nostoc, and Aph-
anizomenon, have been consumed as alternative sources in
supply of human nutrition for centuries, due to their high
protein contents [15, 16]. Spirulina platensis is a widespread
filamentous cyanobacterium in aquaculture, with remarkable
protein content and nutritive value owing to its amino acid
composition [17]. In addition, Spirulina platensis has nutri-
tional properties such as high concentrations of vitamins,
essential fatty acids and minerals [18]. Moreover, the phy-
cobiliproteins in Spirulina platensis have attracted increased
attention for their potential values in food, pharmaceutical

13
Vol.:(0123456789)

1108
and cosmetic industries [19, 20]. Several Spirulina platen-
sis-derived bioactive peptides and various ACE inhibitory
peptides have been identified [21–23]. Different commercial
proteases were used independently or in sequence during the
hydrolysis of Spirulina platensis proteins [21–23]. Shorter
peptides, such as tripeptides and tetrapeptides, which were
believed to be easily absorbed in the intestine, were less
studied. Meanwhile, the toxicity of peptides and the struc-
ture–activity relationships between peptides and the ACE
molecule are still largely unknown. In this study, we aim
to screen novel shorter peptides from Spirulina platensis
hydrolysates with relatively high ACE inhibitory activity
and reveal the binding mode of peptides in the ACE mol-
ecule. First, Spirulina platensis hydrolysates were digested
by thermolysin, and then the peptides were isolated through
size exclusion-high-performance liquid chromatography
(SEC-HPLC) and identified by high performance liquid
chromatography-mass spectrometry (HPLC–MS), there fol-
lowed ACE inhibitory activity detection. Finally, molecular
docking was employed to explore the structure–activity rela-
tionship between inhibitory peptides and the ACE molecule.
Materials and methods
Materials
The Spirulina platensis (SP) powder was bought from
Shanghai Guangyu Biotech Limited Co. (Shanghai, China).
Thermolysin (EC 3.4.24.27) from Geobacillus stearo-
thermophilus, angiotensin-I converting enzyme (ACE;
EC 3.4.15.1) from rabbit lung, and hippuryl-histidyl-leu-
cine (HHL) hydrate were purchased from Sigma–Aldrich
Chemical Co. (Temecula, CA, USA). The synthetic peptides
(purity > 98%) were supplied by Qiangyao Biotech Lim-
ited Co. (Suzhou, China). Acetonitrile of HPLC grade was
obtained from Tidea Reagent, Ltd. (Ohio, USA). All other
chemicals were analytical grade unless otherwise described.
Generation of SP protein hydrolysate (SPH)
SP powder was dispersed in distilled water (3%, w/v) and
subsequently treated by repeated freezing–thawing method.
First, SP powder solution was frozen at about − 20 °C for
several hours, and then thawed at room temperature. After
the third freezing–thawing treatment, the SP samples were
subsequently hydrolyzed by 2.0 wt% of thermolysin at 70 °C
for 3 h. The reaction was ended by heat treatment at 100 °C
for 10 min. The samples were centrifuged at 13,000g for
20 min at 4 °C. The cut-off membranes (5.5 cm discs, Mil-
lipore) with size of 3 kDa were used to fractionate the super-
natant. The ultrafiltration fractions of SPH with molecular
13
European Food Research and Technology (2022) 248:1107–1115
weight (MW) < 3 kDa were designated as SPH-3 and dried
by lyophilization for further analysis.
Isolation of peptides from SPH‑3
The SPH-3 samples were dissolved in deionized (Milli-
Q) water and filtered by Millex-GV (pore size: 0.22 µm).
SEC-HPLC was applied to isolate different peptides as
described with minor modifications [24, 25]. The filtered
samples were injected into a TSK gel G2000 SWXL column
(7.8 × 300 mm, Tosoh, Japan), then eluted with 45% (v/v)
acetonitrile mixed with 0.1% (v/v) trifluoracetic acid (TFA)
at a 1 mL/min flow rate, and monitored at 214 nm. Different
peak fractions were collected for further assay.
HPLC–MS analysis and identification of peptide
sequences
The peak fraction from SPH-3 with high ACE inhibitory
activity was analyzed by HPLC–MS using an ESI-Q-TOF
mass spectrometer (impact HD, Bruker, Germany). The two
mobile phases were as follows: mobile phase A comprised
0.1% formic acid in deionized water, and mobile phase B
was a 0.1% formic acid aqueous solution of acetonitrile.
The mass spectrometry test information was searched for
the corresponding peptides based on the data of SP from the
Uniprot database (https://​www.​unipr​ot.​org/).
ACE inhibitory assay
ACE inhibitory assay was carried out according to the
described method [26] with slight modifications. Two micro-
liters of sample solution were added into 20 µL of ACE (0.2
units/mL), and the mixture was pre-incubated at 37 °C for
5 min. Ten microliters of Hip–His–Leu solution (12.5 mM
in 0.1 M sodium borate buffer containing 400 mM NaCl, pH
8.3) and 33 µL of sodium borate buffer (0.1 M, containing
400 mM NaCl, pH 8.3) were then added into the mixture.
After incubation at 37 °C for 1 h, the reaction was terminated
by adding 100 µL of 1.0 M HCl, and the solution was filtered
through 0.45 µm nylon syringe filter before being analyzed
by reversed-phase high performance liquid chromatography
(RP-HPLC) (Shimadzu, Japan) on a reversed-phase SunFire
­C18 column (4.6 × 250 mm, Waters, USA). The mobile phase
comprised of deionized water:acetonitrile = 50:50 (v/v, with
0.1% TFA) with a flow rate of 0.4 mL/min and the absorb-
ance was monitored at 228 nm. The inhibitory rate (%) was
determined as below:
ACE inhibitory rate (%) =
[( ) ]
Acontrol − Ainhibition ∕Acontrol × 100%,
where Acontrol was the peak area of hippuric acid with the
same volume of the buffer solution instead of the sample,
European Food Research and Technology (2022) 248:1107–1115 1109
and Ainhibition was with the sample. The ­IC50 values (µM) of
different peptides were defined as the sample required to
reach 50% efficiency calculated by five different concentra-
tions of the sample.
In silico prediction of toxicity
The peptides from SPH-3 fraction were evaluated for poten-
tial toxicity in silico using ToxinPred, available at http://​
crdd.​osdd.​net/​ragha​va/​toxin​pred/ [27].
Molecular docking analysis
Molecular docking analysis was carried out to investigate
the interaction between the inhibitory peptides and the ACE
molecule using AutoDock 4.2 (Scripps Research Institute,
La Jolla, CA, USA) with the MGL Tools 1.5.6 package.
Three-dimensional structure file of ACE protein (PDB ID:
1O8A) was derived from the Protein Data Bank (http://w ​ ww.​
rcsb.​org/​pdb/​home/​home.​do). Remove all the water mole-
cules and unwanted substructures before the docking, and
then add the polar hydrogen. The search grid was carried
out as follows: coordinates x 41.678, y 38.308, and z 46.652
with the grid box size of 50 × 50 × 50 Å. The default param-
eters were used if it was not mentioned. The docking model
in which the ligand displays the lowest binding energy in
the binding pocket of protein was chosen as the best model.
PyMOL (http://​www.​pymol.​org/) was used for viewing the
diagrams of the protein–ligand interaction.
Statistical analysis
One-way ANOVA, followed by Duncan’s multiple-range
tests using SPSS Statistics 19.0 (SPSS, Inc., USA) was used
for data analysis with a significance difference (P < 0.05).
The results were expressed as mean ± standard deviation
(SD).
Results and discussion
Amino acid composition analysis of major SP
protein
The major components of SP were crude protein (constitut-
ing 60%–70% of its dry weight) [28], suggesting that SP
is an excellent protein resource for bioactive peptide pro-
duction. Phycobiliproteins are the dominant composition of
SP protein, with colored pigments that act as light anten-
nae in photosynthesis [29, 30]. Previous studies reported
that phycobiliproteins exhibit anti-inflammatory, antioxi-
dant and anticancer properties and are used in the food and
cosmetic industries as a natural dye [20, 31, 32]. As the
main ingredients of SP protein, phycobiliproteins consist
of phycocyanin and allophycocyanin, and both are formed
by two kinds of subunits, α-subunit and β-subunit [33]. The
amino acid compositions of phycobiliproteins were ana-
lyzed (Table 1) by ProtParam Tool, available at https://​web.​
expasy.​org/​protp​aram/, which showed that these four kinds
of subunits contained a relatively high proportion of essen-
tial amino acids, indicating that SP is a promising nutritional
protein source for bioactive peptides generation, in consist-
ent with the previous study [34]. As previously reported,
ACE appears to prefer substrates or competitive inhibitors
that contain hydrophobic amino acid residues, particularly
Val, Leu, and/or aromatic residues (Trp, Tyr, or Phe) at the
C-terminals [35]. As shown in Table 1, hydrophobic amino
acids (Ile, Leu, Val, Pro, Ala) and aromatic amino acids (Tyr,
Trp) were prevalent in phycobiliproteins.
Separation of ACE inhibitory peptides from SP
protein
The variety of proteases can lead to enormous differences
in the bioactivity of peptides. Thermolysin is a low-specific
protease with relatively broad specificity and preferentially
cleaves sites with bulky and aromatic residues (Ile, Leu,
Val, Ala, Met, Phe) in position P1′ [36]. The cleavage of
proteins by thermolysin leads to produce various low MW
peptides with hydrophobic or aromatic terminus, which may
be beneficial to be bound by the ACE molecule. Therefore,
thermolysin was used for SP protein digestion in this study.
Before the enzymatic digestion, repeated freezing–thaw-
ing treatments were employed for a higher efficiency of
phycobiliprotein extraction, considering that phycobilipro-
teins are water-soluble intracellular proteins and assemble
into phycobilisomes, a supramolecular complex [30]. The
SP solution became blue instead of green after pre-treat-
ments, implying that most phycobiliproteins were released
from phycobilisomes. The hydrolysis temperature was set at
70 °C, the optimum temperature for the enzymatic activity
of thermolysin, which can improve the hydrolysis efficiency.
In the meantime, the control groups, with the same volume
of distilled water instead of thermolysin, were incubated at
70 °C for 3 h as well. It has been reported that peptides
with MW < 3 kDa possess high ACE inhibitory activity
[5]. Therefore, a membrane filtration step was further per-
formed to separate different MWs fractions, and fractions
with MW < 3 kDa from both enzymatic samples and control
samples, designated as SPH-3 and SPC-3, were collected,
respectively. The dry weight of SPH-3 after lyophilization
was higher than that of SPC-3, demonstrating that more pep-
tides were released from protein by thermolysin hydrolysis.
The ACE inhibition assays of both SPH-3 and SPC-3 were
examined at a concentration of 5 mg/mL, and the inhibi-
tory rate of SPH-3 was 75.12% ± 1.98%, higher than that
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1110 European Food Research and Technology (2022) 248:1107–1115

Table 1  Amino acid Amino acid Amino acid composition (%)

composition of C-phycocyanin
(C-PC) and allophycocyanin C-PC-α subunit C-PC-β subunit APC-α subunit APC-β subunit
(APC) subunits from SP
Ala 14.80 16.90 11.20 13.70
Arg 4.30 6.40 7.50 5.60
Asn 3.70 4.10 1.90 3.70
Asp 5.60 5.80 5.00 6.20
Cys 1.20 2.30 0.60 1.20
Gln 4.30 2.90 1.20 3.10
Glu 4.90 4.70 9.30 3.70
Gly 8.00 6.40 9.90 8.10
His 0.60 0.00 0.00 0.00
Ile 6.80 5.20 7.50 7.50
Leu 8.00 8.10 6.80 8.70
Lys 5.60 2.30 3.70 5.00
Met 2.50 3.50 3.70 3.10
Phe 3.10 2.90 1.90 1.20
Pro 3.10 2.90 3.10 1.90
Ser 7.40 9.30 6.80 6.80
Thr 6.20 5.80 6.20 6.80
Trp 0.60 0.00 0.00 0.00
Tyr 6.80 2.90 5.00 7.50
Val 2.50 7.60 8.70 6.20
Total hydrophobic amino acids 33.40 39.00 36.10 33.60
Total essential amino acids 35.90 35.40 38.50 38.50

of SPC-3, which was only 34.17% ± 2.35%, indicating the
efficiency of enzymatic digestion.
SPH-3 sample was further separated by SEC-HPLC
using a TSK gel G2000 SWXL column, and five major
peaks (P1-P5) at 214 nm (Fig. 1A) were detected. Differ-
ent peak fractions at a concentration of 5 mg/mL possessed
distinct inhibitory activity against ACE (Fig. 1B). Fractions
P1 and P2 exhibited lower inhibitory activity than SPH-3,
and Fractions P3 and P5 showed no significant difference
with SPH-3. Among them, Fraction P4 exhibited the high-
est inhibitory activity (81.85%), suggesting the enrichment
of inhibitory peptides. These results demonstrated that

Fig. 1  Chromatogram of SPH-3 fraction and ACE inhibitory activity of different peak fractions. A Purification from SPH-3 by SEC-HPLC. B
ACE inhibitory activity of each peak fraction. Different letters in the same line indicate significant differences (P < 0.05)

13
European Food Research and Technology (2022) 248:1107–1115 1111
Fraction P4 possessed the most potent ACE inhibitory activ-
ity among the five peaks and thus was selected for further
identification.
Mass identification of ACE inhibitory peptides
The components of Fraction P4 were further identified by
HPLC–MS. The amino acid sequences of peptides from SP
were searched in the Uniprot database (http://​www.​unipr​ot.​
org/). According to previous studies, most ACE inhibitory
peptides are small peptides possessing 2–12 residues, which
may be bound in the ACE active site more easily and thus
assert inhibitory activity [5]. In our study, most detected
peptides contained less than five amino acids. The top eight
abundant peptides are listed in Table 2, among which five
novel peptides, including LRDMEI, LRETY, LGVP, FDY,
and VTY, were determined based on the BIOPEP-UWM
database (http://​www.​uwm.​edu.​pl/​bioch​emia/​index.​php/​en/​
biopep). The other three peptides, LDY, IDA, and IQK, were
reported as either dependent ACE inhibitory peptides or par-
tial sequence of longer ACE inhibitory peptides. Moreover,
sequence alignments revealed that the sequences of these
eight peptides can be matched in the primary structure of
different subunits of phycobiliproteins, indicating that phy-
cobiliproteins may be the main source of bioactive peptides
in SP. This was detected in the enzymatic digestion of other
phycobiliprotein-contained algae protein [37].
The mass spectra of eight peptides are shown in Fig. 2
and the m/zs of eight peptides are summarized in Table 2.
Several studies focused on SP-derived bioactive peptides,
and different proteases were tried in the enzymatic diges-
tion in vitro, such as papain (EC 3.4.22.2), alcalase (EC
3.4.21.14), flavourzyme (EC 3.4.11.1), pepsin (EC 3.4.23.1),
trypsin (EC 3.4.21.4), and neutrase (EC 3.4.24.4) [21–23].
Due to the cleavage specificity of protease, the identified
ACE inhibitory peptides were commonly different. In a simi-
lar study, Anekthanakul et al. employed in gel digestion with
thermolysin in combination with in-silico based digestion to
screen bioactive peptides from SP, and five longer candidate
peptides were finally chosen [21]. However, none of these
Table 2  Information of eight Peptide sequence
identified peptides from SP
LDY
FDY
LRDMEI
IDA
LRETY
IQK
VTY
LGVP
peptides were found in our study, indicating that the peptide
profile of enzymatic hydrolysates from SP may be affected
by enzyme hydrolysis conditions.
Potential toxicity of novel inhibitory peptides
Peptides have numerous advantages over small molecules,
including high biological activity, high specificity, low
production cost, and high penetration. However, toxicity
remains to be the main concern in the development of pep-
tides [28]. The potential toxicity of protein and peptides
can adversely affect human health and clearly be concerned
when used as functional food ingredients or consumed as
nutraceuticals. The in-silico tools for initial prediction of
toxicity has been widely considered, due to the complex
and time-causing features of in vivo and in vitro evalua-
tion system. In this study, in silico toxicity assessment of
the eight identified peptides was conducted using Toxin-
Pred with two modules, SVM and Motif (Table 2), and the
results suggested that none of these peptides was predicted
to be toxic. The toxicity assessment of peptide is necessary
for its further application, especially in developing peptide-
based functional foods and clinical treatments. Melittin, a
significant peptide component of bee venom, was considered
to be an appealing candidate for cancer treatment. However,
its application in functional foods and pharmaceuticals has
been hampered by a number of difficulties, including non-
specific cytotoxicity [41]. Our results were consistent with
the previous research that ACE inhibitory peptide PTGN-
PLSP obtained from phycocyanin was also non-toxic by
ToxinPrep analysis [23].
ACE inhibitory assay in vitro
Two peptides, Val–Thr–Tyr (VTY) and Leu–Gly–Val–Pro
(LGVP), were chosen and synthesized for further analysis.
The ACE inhibitory activities of VTY and LGVP in vitro
were examined, and the results showed that VTY and LGVP
were with the I­C50 values of 23.39  μiM and 45.76  μM,
respectively, exhibiting relatively high inhibitory effects.
No. of amino acid Theoretical Observed molecular ion, Toxin prediction
residues mass (Da) m/z (charge)
3 409.18 410.1915 Non-toxin
3 443.17 444.1766 Non-toxin
6 775.39 776.3920 Non-toxin
3 317.16 318.1797 Non-toxin
5 680.35 681.3540 Non-toxin
3 387.25 388.2538 Non-toxin
3 381.19 382.1962 Non-toxin
4 384.24 385.2436 Non-toxin
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1112 European Food Research and Technology (2022) 248:1107–1115

Fig. 2  Mass spectra of peptides identified by HPLC–MS. A LDY, B FDY, C LRDMEI, D IDA, E LRETY, F IQK, G VTY, H LGVP

13
European Food Research and Technology (2022) 248:1107–1115 1113
According to the previous study [38], the composition of
the C- and N-terminal residues of an ACE inhibitory peptide
also had a significant effect on ACE inhibition rate, with
high inhibitory activity when the C-terminal residue was
Tyr, Phe, Pro, Trp, or Leu and the N-terminal residue was
a hydrophobic aliphatic branched-chain amino acid such as
Leu, Ile, Ala, or Met [38]. This might explain the potent
ACE inhibitory activity of VTY and LGVP, which con-
sisted of tyrosine and proline in the C-terminal, as well as
hydrophobic amino acid residues valine and leucine in the
N-terminal, respectively.
Molecular docking between peptides and ACE
molecule
Molecular docking was performed to estimate the bind-
ing modes between peptides and ACE using AutoDock
4.2 (http://​autod​ock.​scrip​ps.​edu/). The best results were
obtained for VTY and LGVP at the ACE active site in
the presence of Zn (II) (Fig. 3), with binding energies of
− 6.48  kcal/mol and − 5.57  kcal/mol, respectively. As
Fig. 3  Local overview of the
best ranked docking models of
VTY (A) and LGVP (B) bind-
ing with ACE (PDB: 1O8A)
at the ACE catalytic site. The
peptides VTY and LGVP are
shown as sticks colored in yel-
low. ACE residues involved in
binding peptides are shown as
sticks colored in magenta. The
possible hydrogen bonds are
represented by dashed lines
shown in Fig. 3, the interactions between docked peptides
and enzymes were analyzed using PyMOL. The binding of
VTY and LGVP mainly depends on hydrogen bonds formed
between ACE residues and the ligands. For VTY binding,
residues Gln281, His353, Ala354, Glu384, His513, Tyr520,
and Tyr523 can form hydrogen bonds with VTY; the N-ter-
minal valine residue participates in coordinating Z­ n2+; in
particular, two aromatic residues, Phe457 and Phe527, may
form pi–pi stacking interactions with the side chain of the
C-terminal tyrosine residue (Fig. 3A). Residues involved in
binding LGVP are much less than that involved in bind-
ing VTY (Fig. 3). Residues Tyr360, Ala354, His387, and
Tyr523 participate in binding LGVP through hydrogen
bonds. The N-terminal leucine residue also participates in
coordinating ­Zn2+ (Fig. 3B).
It has been reported that three main active site pockets
are involved in the ACE-ligand interaction: the S1 pocket
includes three residues, Ala354, Glu384, and Tyr523;
the S2 pocket includes Gln281, His353, Lys511, His513,
and Tyr520; and the S1′ pocket only includes the residue
Glu162 [39, 40]. Moreover, other key amino acid residues,
13

1114
namely Glu411, Glu162, His383, and His387, were also
important for the ACE activity [41]. Hydrogen bond inter-
actions play an irreplaceable role in stabilizing the struc-
ture of the complex as well as the ACE reaction [40]. As
shown in Fig. 3, VTY is mainly bound in S1 (Ala354,
Glu384, and Tyr523) and S2 pockets (Gln281, His513,
and Tyr520) via hydrogen bonds, while LGVP only inter-
acts with Ala354 and Tyr523 of the S1 pocket, which may
explain the lower I­ C50 value of LGVP. Previous research
revealed that some ACE inhibitory peptides, based on a
non-competitive inhibition mechanism, could not interact
with the main active site of ACE [8]. Therefore, VTY and
LGVP may employ different strategies in the interaction
with ACE.
Conclusions
In this study, Spirulina platensis proteins were hydro-
lyzed using thermolysin. The component with the highest
inhibitory activity of ACE was obtained by a series of
purification steps, including membrane filtration and SEC-
HPLC. Five novel peptides were identified by HPLC–MS,
among which, VTY and LGVP were with relatively low
­IC50 values against ACE (23.39 μM and 45.76 μM, respec-
tively). The results of the docking simulation investigation
suggested that VTY displayed a strong binding power to
the active pocket S1 (Ala354, Glu384, and Tyr523) and
S2 (Gln281, His513, and Tyr520) of ACE via hydrogen
bonds, and LGVP only interacted with the S1 pocket
(Ala354 and Tyr523). In general, it seemed to exert antihy-
pertension through inhibiting the activity of ACE, which
was similar to the antihypertensive mechanism of capto-
pril. Structure–activity relationship indicated that the pres-
ence of specific amino acid residues in the sequence, such
as hydrophobic and aromatic residues, may be responsi-
ble for the ACE inhibitory activity of these two peptides.
These results together indicated that the potential possibil-
ity of VTY and LGVP as alternatives of synthetic drugs to
treat hypertension. Certainly, we will further investigate
whether these two identified peptides had antihypertensive
effects and the bioavailability and mechanism of action
in vivo.
Acknowledgements  This study was supported by the Natural Science
Foundation of Shandong (No. ZR2017LD013) and the Key Research
and Development Program of Shandong Province (No. 2019YYSP024,
2019GNC106154).
Author contributions  NZ and SY conceived and designed the study.
NZ, FL, TZ, CL, and LZ worked for the experiment. NZ and SY were
major contributors in writing the manuscript. All authors read and
approved the final manuscript.
13
European Food Research and Technology (2022) 248:1107–1115
Declarations 
Conflict of interest The authors declare that the research was con-
ducted in the absence of any commercial or financial relationships that
could be construed as a potential conflict of interest.
Compliance with ethics requirements  We all comply with Ethics re-
quirements of the journal “European Food Research and Technology”.
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