Subido por ecef1212

Franci

Anuncio
Journal of Chromatography B 1186 (2021) 122990
Contents lists available at ScienceDirect
Journal of Chromatography B
journal homepage: www.elsevier.com/locate/jchromb
Purification, identification and molecular mechanism of dipeptidyl
peptidase IV inhibitory peptides from discarded shrimp (Penaeus
vannamei) head
Xi Xiang a, Meng Lang a, Yan Li a, Xia Zhao a, Huimin Sun a, Weiwei Jiang a, Ling Ni a,
Yishan Song a, b, *
a
b
College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China
National R&D Branch Center for Freshwater Aquatic Products Processing Technology (Shanghai), Shanghai 201306, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
DPP-IV inhibitory peptides
Purification
Identification
P. vannamei head
Molecular docking
DPP-IV plays a key role for regulation of glucose metabolism in the body. The object of this study was to obtain
DPP-IV inhibitors from discarded but protein-rich Penaeus vannamei (P. vannamei) head, and to explore the
potential mechanism between DPP-IV and its inhibitors. P. vannamei head protein was hydrolyzed by five food
grade proteases, respectively. The animal protease hydrolysate showed the highest inhibitory active. Then the
hydrolysate was sequentially separated by ultrafiltration, gel filtration chromatography and reversed phase highperformance liquid chromatography (RP-HPLC), the peptides sequences were identified by LC-MS/MS and four
potential peptides YPGE, VPW, HPLY, YATP showed superior DPP-IV inhibitory activity. Meanwhile, molecular
docking effectively explored their mechanism through formed hydrogen bonds and hydrophobic regions. The
four peptides showed better DPP-IV inhibitory activity stability with heating treatment, pH (1–10) treatment,
and in vitro gastrointestinal digestion. Our results demonstrated that the protein hydrolysate from discarded
P. vannamei head can be considered as a promising natural source of DPP-IV inhibitor for helping to improve
glycaemic control in Type 2 diabetes.
1. Introduction
Diabetes mellitus is a metabolic disease characterized by high blood
sugar and caused by impaired insulin secretion or defective function
with insulin resistance [1], and long-term hyperglycemia will lead to
chronic damage and dysfunction of various tissues [2]. However, the
prevalence of diabetes has increased in the recent years due to the
improvement of living standards, the appearance of unhealthy lifestyle
and the change of dietary structure [3,4], especially the type 2, which
accounts for over 90% of diabetics [5]. Diabetes have already become a
huge socio-economic challenge and an important health problem
worldwide in the 21st century [6,7]. Related researches illustrate that
there are more than 400 million people nowadays diagnosed with dia­
betes [8], meanwhile, the forecasts according to International Diabetes
Federation (IDF) indicate that almost 700 million people are likely to
suffer from diabetes by 2045 [9].
Currently, it is the most common method for diabetes treatment to
combine lifestyle modification with some hypoglycemic drugs, but this
method is difficult to achieve the desired effect, and ultimately may
requires exogenous insulin [10]. Therefore, current research focuses on
finding simpler and more effective anti-diabetic methods, for instance,
modulating molecular targets of diabetes, which involves regulating the
activity of α-amylase, α-glucosidase, and dipeptidyl peptidase IV[11].
Among them, DPP-IV is considered to be the greatest potential from its
mechanism of action for the treatment of diabetes.
DPP-IV, a transmembrane serine protease, is distributed widely in
plasma, kidney, intestinal villi and plasma cells, and can be found it in
almost all human cell [12,13]. The function of DPP-IV is to inactivate
certain proteins and peptides in the body [14], such as glucagon-like
peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide
(GIP) [15]. GLP-1 and GIP, as incretin hormones, decrease the blood
glucose concentration effectively in the body by promoting insulin
secretion, inhibiting the release of glucagon, and enhancing pancreatic β
cells proliferation [16]. The concentrations of GLP-1 and GLP are sup­
posed to increase and last for a while after food intake, but rapidly
decrease and their half-lives are only 1–2 min due to the degradation by
* Corresponding author at: College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China.
E-mail address: [email protected] (Y. Song).
https://doi.org/10.1016/j.jchromb.2021.122990
Received 20 July 2021; Received in revised form 7 October 2021; Accepted 11 October 2021
Available online 28 October 2021
1570-0232/© 2021 Elsevier B.V. All rights reserved.
X. Xiang et al.
Journal of Chromatography B 1186 (2021) 122990
DPP-IV [17].
DPP-IV inhibitors can prevent incretins cleavage and increase the
half-life of the active hormones, which increasingly attracted re­
searchers’ attention [18]. Currently, inhibitor types can be divided into
two broad categories, including synthetic inhibitors and natural ones.
Those synthetic inhibitors, such as Vildagliptin, Sitagliptin, Linagliptin,
significantly control blood glucose in a short time, but a series of un­
desirable side effects may occur with time, including gastrointestinal
and hepatic disorders [19]. These undesired effects have prompted the
current research favor the other alternative inhibitors, that is, extracting
bioactive peptide with DPP-IV inhibitory activity from food-derived
sources, which can contribute to a positive therapeutic response with
fewer undesired effects [6].
Presently, many studies have acquired DPP-IV inhibitory peptides
from various food-derived plants, such as corn [20], rice [21], pumpkin
[22], and animals such as tuna [23], ham [24], Antarctic krill [13],
chicken feet [25], as well as milk proteins [26]. Moreover, we can
consider using plentiful and available marine resources underutilized in
the food industry as raw materials, which may be potential sources of
bioactive peptides [27].
Shrimp plays an important role in the world aquatic product market,
as early as 2013, the annual output exceeded 4.3 million tons, valued at
more than 22 billion US dollars [28]. P. vannamei, one of the main
species in shrimp farming worldwide, accounts for nearly 90% and 78%
of shrimp aquaculture in the West and Asia respectively [29]. The pro­
duction of P. vannamei has raised significantly in the past few decades
from 1.31 to 3.75 million metric tonnes by 2018, and the production is
expected to reach 4 million metric tonnes by 2021 [30]. Although
P. vannamei brings huge commercial and ecological values to society, it
produces a large amount of by-products during processing or eating. For
instance, shrimp head contains 17.3% protein (moisture 74.95%) and
3% fat, which makes it a good choice as the raw material with high
protein and low fat. The traditional treatment method is simplely pro­
cessed as fodder or directly discard, which will not only lead to massive
resource waste, but also cause environmental pollution. Hence, new
methods need to be found to solve those problems.
Present research on P. vannamei focuses on practical economics,
mainly regarding their breeding, transportation and storage. There is
little research about extracting biopeptides from P. vannamei, let alone
obtaining DPP-IV inhibitory peptides from discarded P. vannamei head.
Fortunately, some other shrimp species have been studied to obtain
biopeptides successfully, including antimicrobial peptide [31], ACE-I
inhibitory peptide [32], as well as DPP-IV inhibitory peptides [13].
Therefore, we hypothesized that P. vannamei head would be a potent
source of bioactive peptides with antidiabetic effect.
The objectives of the present study were to generate protein hydro­
lysates with DPP-IV inhibitory ability from discarded P. vannamei head
with different enzymes and to purify and identify potential peptide se­
quences. Besides, the mechanism between DPP-IV and the prepared
DPP-IV inhibitory peptides was explored by molecular docking.
were purchased from Merck Company, Ltd. (Darmstadt, Germany). The
following reagents were purchased from Sigma-Aldrich (Shanghai,
China): O-Phthalaldehyde (OPA), sodium dodecyl sulfate (SDS),
DL–dithiothreitol (DTT).
2.2. P. vannamei heads protein hydrolysates
The protein content in fresh shrimp heads was determined to be
17.3% (moisture 74.95%), this is more in line with the industrial pro­
duction process of extracting peptides from high-protein substances,
thus, the fresh shrimp heads were used for hydrolysis directly without
protein extraction.
In order to choose suitable protease, hydrolysis was conducted using
five food grade protease selections of enzyme based on results of pre­
liminary experiments. Briefly, the different enzymolysis temperature
and pH conditions of each protease were as follows: Flavor protease (pH
7.0, 50 ◦ C), papain (pH 7.5, 45 ◦ C), alkaline protease (pH 9.0, 50 ◦ C),
animal protease (pH 7.5, 50 ◦ C) and compound protease (pH 7.0, 50 ◦ C),
the pH of each enzymatic procedure was adjusted to the working value
of the selected enzyme using 1.0 M NaOH and HCl. All of the substrateliquid (W/V) ratio and the enzyme-substrate (E/S) ratio were 1:5 and
3000 U/g. The duration of all hydrolysis reactions was 5 h. Furthermore,
Hydrolysis was stopped by heating at 100 ◦ C for 15 min. The optimized
conditions after the final choice of using animal protease were the
substrate-liquid (W/V) ratio and the enzyme-substrate (E/S) ratio were
8.81:1 and 2100 U/g, the enzymolysis time was adjusted to 4.3 h. The
selected index is the inhibition rate of DPP-IV. The degree of hydrolysis
and the molecular weight distribution of the enzyme hydrolysate are
also discussed.
2.3. Ultrafiltration
The portion of the supernatant containing the target peptides was
passed through different ultrafiltration membranes (Millipore Corpora­
tion, Bedford, MA, USA) with molecular weight cutoffs. The fractions
were collected at different molecular weight ranges containing >5000
Da, between 5000 Da and 3000 Da, 3000–1000 Da, and <1000 Da. Four
fractions collected were concentrated, lyophilized and stored at − 20 ◦ C
for further analysis.
2.4. Gel filtration chromatography
The ultrafiltered fractions with the highest DPP-IV inhibitory activity
were dissolved in deionized water (20 mg/mL). Subsequently, 5 mL of
the sample was injected into a Sephadex G-15 gel column (1.6 cm × 50
cm) for elution using deionized water at 0.5 mL/min. The fractions were
collected in 2 mL per tube and measured at 254 nm to determine the
absorbance of the samples eluted curves by a protein purifier system
(HDB-7, Huxi Analysis Instrument Factory Co., Ltd, Shanghai, China).
The peptides were fractionated as depicted in the five peaks obtained.
The different fractions were collected, lyophilized, and then stored at
− 20 ◦ C for further analysis.
2. Materials and methods
2.1. Materials and chemicals
2.5. Analytical and preparative reverse-phase high-performance liquid
chromatography (RP-HPLC)
P. vannamei were purchased from Yangdong food market (Shanghai,
China), and the species was determined to be Number: SC 2055–2006
according to the “Aquatic Products Standards of the People’s Republic of
China”. The shrimp head obtained from these live shrimps were kept at
− 80 ◦ C until use.
DPP-IV inhibitor screening assay kit was purchased from Sigma
Chemicals Co. Ltd. (St. Louis, MO, USA). Flavor protease (1.98 × 104 U/
g), papain (2.28 × 104 U/g), alkaline protease (7.34 × 104 U/g), animal
protease (6.35 × 104 U/g) and compound protease (5.73 × 104 U/g)
were from Nanning Dongheng Huadao Biotechnology Co., Ltd. (Guangxi
Province, China). HPLC-grade acetonitrile and trifluoroacetic acid (TFA)
The highest inhibitory fraction separated by the gel filtration column
was lyophilized and resuspended in ultrapure water (10 mg/mL). The
sample was analyzed using the Waters e2695 separations module (Wa­
ters Technologies, Milford, MA, USA) equipped with a detector (Waters
2489 UV/Vis Detector). The sample (10 μL) was injected into a C18
column (Waters SunFire®, 5 μm, 4.6 mm × 250 mm). The elution pro­
tocol was performed according to previous experience [33] with a slight
modification: 0.1% (v/v) TFA in ultrapure water (A) and 0.1% TFA in
acetonitrile (ACN) (B). The gradient sequence was as follows: 20% B
from 0 to 5 min, 20–45% B from 5 to 35 min, and 45–20% B from 35 to
2
X. Xiang et al.
Journal of Chromatography B 1186 (2021) 122990
45 min, at a flow rate of 0.5 mL/min. The absorbance of the fraction was
monitored at 214 nm; subsequently, the sample was prepared on the
Elitehplc P230 preparative RP–HPLC (Dalian Elitehplc Analytical In­
strument Co., Ltd, Dalian, China). The samples (2 mL) were injected into
a C18 column (Waters SunFire® Prep C18 OBDTM, 5 μm, 19 mm × 150
mm). The elution conditions were slightly changed based on the
analytical RP-HPLC. All the peaks were pooled, frozen and lyophilized
immediately for further analysis.
group). The half-maximal inhibitory concentration (IC50) values were
calculated with SPSS software based on the dose–response between
logarithm of the sample concentration (mg/mL) and DPP-IV inhibitory
activity (%).
2.9. Peptide identification
The fraction gathered was analyzed by LC-MS/MS, which exhibited
the highest DPP-IV inhibitory activity after purification. After desalting
and pre-treatments, the lyophilized peptide samples were reconstituted
in 40 μL of 0.1% TFA solution. Agilent 1100 instrumentation was used
and equipped with a 75um × 150 mm RP-C18 Column. The flow rate of
the separation process was 250 nl/min. The separated samples were
analyzed by Q Exactive mass spectrometer (Thermo Fisher), the condi­
tions were as follows: analysis time: 60 min; detection method: positive
ion, precursor ion scan range: 300–1800 m/z, resolution of primary mass
spectrometer: 70,000 (m/z is 200), AGC target: 1e6, IT value: 10 ms,
Number of scan ranges: 1, and dynamic exclusion: 20.0 s.
Ten fragmentation maps were collected (MS2 scan) after each full
scan (full scan). MS2 activation type: HCD, isolation window: 1.6 m/z,
secondary resolution: 17,500 (m/z was 200), micro scan: 1, secondary
maximum IT value: 60 ms, normalized collision energy: 27 eV, Underfill
rate: 0.1%.
The resulting of MS files was processed using the MaxQuant software
(version 1.5.5.1). The processed MGF files were searched against the
UniProt P. vannamei_26335_20210104 (containing 26,335 sequences,
downloaded on Jan 4, 2021) without specifying enzyme cleavage rules.
The search parameters were set as follows: ±20 ppm for peptide mass
tolerance, 0.1 Da for MS/MS tolerance, 2 for maximum missed cleavage
(with an allowance for 2 missed cleavages). Variable modification:
Oxidation (M). Label-free peptide quantification based on extracted ion
chromatograms and spectral counts and validation was performed in the
MaxQuant software. The cutoff value of global false discovery rate
(FDR) for peptide identification was set to 0.01.
2.6. Determination of degree of hydrolysis (DH)
The degree of hydrolysis was analyzed by the OPA method following
a previous report with slight modifications [34]. The OPA reagent (200
mL) was prepared as follows: 7.620 g sodium tetraborate, 200 mg SDS,
and 176 mg DTT were dissolved in 150 mL distilled water. After that,
exactly 160 mg OPA was dissolved in 4 mL anhydrous ethanol. Then,
distilled water was added to obtain a final of 200 mL; the final reagent
was protected from light. DH was calculated by the following formula:
DH =
h
× 100%
ht0 t
where h was calculated based on the OD of serine standard (100 mg
serine was dissolved in 1L distilled water) and samples. h tot = 7.84
mequiv/g.
2.7. Determination of molecular-weight distribution
The molecular-weight distribution of the P. vannamei head hydro­
lysate were estimated by high-performance size-exclusion chromatog­
raphy (SEC-HPLC) using Waters 2695–2489 instrumentation equipped
with a TSK gel 2000 SWXL column (300 × 7.8 mm, Tosoh, Tokyo,
Japan).
The lyophilized powder of the enzymatic hydrolysis solution was
resuspended in ultrapure water to a concentration of 10 mg/L; 0.22 μm
hydrophilic membrane is required before injection; isocratic elution was
at a flow rate of 0.5 mL/min and 30 ◦ C and monitored at 220 nm. The
composition of solvent was Acetonitrile, ultrapure water and trifluoro­
acetic acid (45:55:0.1). A molecular-weight calibration curve was pre­
pared from the average retention times of the following standards:
bovine serum albumin (MW = 67000 Da), cytochrome c (MW = 12500
Da), rapeseed peptide (MW = 1158.57 Da), glutathione (MW = 307.32
Da), and glycine (MW = 75 Da; Sigma Company, St. Louis, MO).
2.10. Peptide synthesis
Peptides selected from the LC-MS/MS identification results were
synthesized by DGpeptides Co., Ltd, Hangzhou, China. The purity of the
synthesized peptides is higher than 95%, analyzed by RP-HPLC-MS/MS.
2.11. Molecular docking
The inhibitory mechanism of identified DPP-IV inhibitory peptides
and DPP-IV was studied by the molecular docking method. The receptor
DPP-IV (PDBID:5Y7H) here come from Protein Data Bank (http://www.
rcsb.org/pdb). Software PyMOL 2.3.4 was used to dehydrate and deli­
gand. Auto Dock Tools software (version 4.2) was performed to modify
the receptor protein by hydrogenation and free radical balancing. Af­
finity (grid) maps were generated using the Autogrid program with a
0.375 Å spacing. DPP-IV was set to rigid molecular, whereas the torsion,
position and orientation of peptides were set randomly. DPP-IV inhibi­
tory peptides and DPP-IV protein model were docked by Auto Dock Vina
1.1.2 software.
2.8. Assay for DPP- IV inhibitory activity
DPP-IV inhibitory activity was measured using DPP-IV inhibitor
screening assay kit (Sigma Chemicals Co. Ltd, St. Louis, Mo, USA)
described in previous literature [13] with some modifications. Human
recombinant DPP-IV enzyme and DPP-IV substrate (Gly-pro-AMC/AFC)
were included in the DPP (IV) inhibitor screening assay kit. Briefly, the
freeze-dried powder sample was dissolved in the buffer for dilution.
Sample solution (10 μL), DPP-IV solution (50 μL) was added to a 96wells plate and incubated at 37 ◦ C for 10 min, following adding 25 μL
substrate solution to all the wells and incubated at 37 ◦ C for 15 min.
After that, read the plate with a microplate reader (Varioskan Flash,
Thermo Fish Scientific Co., Massachusetts, USA). The fluorescence was
measured at an excitation wavelength of 360 nm and an emission
wavelength of 460 nm. All steps were kept in a dark environment to
ensure that the substrate is not decomposed. The inhibition activity was
calculated by the following formula:
DPP − IV inhibition activity (%) =
2.12. Stability of DPP-IV inhibitory peptides activity
The stability of the four synthetic DPP-IV inhibitory peptides activity
against simulated gastrointestinal digestion, thermal and pH treatments
was measured following a previous report with minor modifications
[35]. Briefly, for stability against simulated gastrointestinal digestion,
peptides (20 mg) were dissolved in 10 mL of distilled water and the pH
were adjusted to 2.0 with 1 M HCl. 2% (w/w) pancreatin was added
followed by incubation in a shaking incubator for 120 min at 37 ◦ C. After
simulate gastric digestion, the pH of the mixture was adjusted to 7.5
with 1 M NaOH, 2% (w/w) trypsin was added and incubated in a
A− B
× 100%
B
where A represented solution with no DPP-IV inhibitor (blank group). B
represented solution containing sample or DPP-IV inhibitor (the sample
3
X. Xiang et al.
Journal of Chromatography B 1186 (2021) 122990
shaking incubator for 120 min at 37 ◦ C to simulate intestinal digestion.
Samples were collected at 30, 60, 120, 150, 180 and 240 min during in
vitro digestion. After sampling, the obtained samples were quickly
placed at 100 ◦ C for 15 min to inactive relevant enzymes. The sample
without any treatment was the control and the DPP-IV inhibitory ac­
tivity was 100%.
For thermal stability of DPP-IV inhibitory peptides activity, the four
peptides solution (10 mL) with the concentration of 0.2 mg/mL were
heated for 30 min at 37, 50, 60, 70, 80, 90, 100 or 121 ◦ C, respectively.
After, these samples were immediately cooled in iced water. The sample
without heat treatment (25 ◦ C) was used as the control. For pH stability
of DPP-IV inhibitory peptides activity, the four peptides solution (10
mL) with the concentration of 0.2 mg/mL were treated for 30 min at
room temperature and pH 1.0, 3.0, 5.0, 7.0, 9.0 and 11.0. The pH of the
treated samples was immediately adjusted to 7.0. The sample without
pH treatment (25 ◦ C) was used as the control.
All of samples collected were concentrated, lyophilized and stored at
− 20 ◦ C for further analysis. The residual DPP-IV inhibitory activity was
determined and expressed as the activity (%) relative to that without any
treatment (control, 100%).
DPP-IV inhibitory activity was indeed lower than that of Napin treated
with alcalase and trypsin. They concluded that the flavor protease failed
to cleave basic amino acids accurately, which accounted for the majority
of amino acid residues in DPP-IV-inhibitory peptides [5].
Many previous articles pointed out that DPP-IV inhibitory peptides
were related to molecular weight of peptides, especially the peptides
composed of 2 to 10 amino acid residues. Harnedy-Rothwell et al. suc­
cessfully extracted a pentapeptide IPVDM from a boarfish (Capros aper)
protein hydrolysate and its DPP-IV half maximal inhibitory concentra­
tion value was 21.72 ± 1.08 µM in a conventional in vitro incubation
system [36]. Nongonierma et al. prepared two DPP-IV inhibitory pep­
tides from camel milk protein hydrolysates, LPVP and MPVQA, with the
IC50 values of 87.0 ± 3.2 and 93.3 ± 8.0 µM, respectively [37]. In this
study, the molecular weight distribution of the treated hydrolysates was
showed in Fig. 1b. The proportion of MWs less than 1 kDa in all hy­
drolysates reached more than 85%, but their DPP-IV inhibitory activity
varied. This phenomenon indicated that not all low molecular weight
peptides had DPP-IV inhibitory activity. Therefore, the choice of en­
zymes was extremely important for the preparation of hypoglycemic
peptides, because enzymes determined the structure and type of low
molecular weight peptides prepared the same raw material. These re­
sults confirmed that the activity of DPP-IV inhibitory peptides was
influenced by the protease used in the process of enzymatic hydrolysis,
rather than the DH value or the content of low molecular weight
peptides.
2.13. Statistical analysis
Data were expressed as the mean ± standard deviations from the
triplicates. Differences between the mean values of triplicate groups
were analyzed by one-way analysis of variance (ANOVA). The statistical
analysis was performed using SPSS 10.0 software (version 22, SPSS Inc.,
Chicago, IL, USA), and the significant difference was determined with a
95% confidence interval (P < 0.05).
3.2. Purification of DPP-IV inhibitory peptides from P. vannamei head
hydrolysate
Ultrafiltration was a common separation technique used to separate
protein hydrolysates based on molecular weight. P. vannamei head hy­
drolysate was separated into four fractions with three different ultra­
filtration members (5000 Da, 3000 Da, 1000 Da). The DPP-IV inhibition
activity of each fraction at a concentration of 10 mg/mL was presented
in Fig. 2a, the DPP-IV inhibition activity of P4 (MW < 1000 Da, 66.42 ±
1.75%) was significantly higher than those of P1 (MW > 5000 Da, 42.78
± 1.12%), P2 (MW = 5000–3000 Da, 42.88 ± 0.87 %), P3(MW =
3000–1000 Da, 58.43 ± 1.56%) and hydrolysate (53.41 ± 2.92%) (p <
0.05). The result was similar to that of many pervious articles, for
instance, Xu et al. found that all the fractions with molecular weight less
than 1 kDa showed the highest DPP-IV inhibitory activity after the napin
treated by five enzyme combinations respectively [5]. It can be
confirmed that DPP-IV inhibitory peptides are related to low molecular
weight peptides again. To obtain purified peptides, P4 was selected to
perform further separation by gel column chromatography. P4 was
separated using Sephadex G-15 to get DPP–IV inhibitory peptides. As
shown in Fig. 2b, five fractions (P4-1, P4-2, P4-3, P4-4 and P4-5) were
3. Results and discussion
3.1. DPP-IV inhibitory activity, degree of hydrolysis, molecular weight
distribution of the hydrolysate of P. vannamei head
The inhibitory activity of DPP-IV is the most favorable evidence for
the evaluation of hypoglycemic effect, and the degree of hydrolysis is
one of the important indexes to assess the effect of enzymatic hydrolysis
in industry. In Fig. 1a, under the animal protease condition, the hy­
drolysate derived from P. vannamei heads showed the strongest DPP-IVinhibiting activity (63.59 ± 2.26%, 20 mg/mL). Fig. 1a. also showed
that the degree of hydrolysis of P. vannamei head hydrolysate under
animal protease was 18.55 ± 0.35% and lower than compound protease
and papain treatment, but this did not affect the magnitude of DPP-IV
inhibitory activity. This phenomenon was parallel to previous studies,
for example, Xu et al. treated Napin protein with Alcalase and fla­
vourzyme and obtained the highest DH value of 20.57 ± 1.87%, but its
Fig. 1. Inhibitory activity (%) and DH (%) of hydrolysates under different enzyme action (a) and molecular weight distribution of different hydrolysates (b).
4
X. Xiang et al.
Journal of Chromatography B 1186 (2021) 122990
Fig. 2. The IC50 value of each fraction after ultrafiltration (a); Sephadex G-15 gel chromatogram (b) and the IC50 value of each fraction (P4-1, P4-2, P4-3, P4-4, P4-5
and P4-2) (c).
obtained and collected separately. The IC50 value of each peak was
measured and demonstrated shown in Fig. 2c. Among them, the fraction
of P4-1 exhibited the best inhibitory activity with the lowest IC50 value
(3.60 ± 0.54 mg/mL), which was lower than those of P4-2 (4.34 ± 0.46
mg/mL), P4-3(6.38 ± 0.30 mg/mL), P4-4(5.28 ± 0.37 mg/mL), P4-5
(4.29 ± 0.22 mg/mL) and P4(7.90 ± 0.54 mg/mL). The separation de­
gree of the peaks in Fig. 2b and the significant difference between the
IC50 values of P4 and P4-1(P < 0.05) indicated that the highest DPP-IV
inhibitory fraction was separated. However, the fractions separated
from gel chromatography usually contained many peptides with similar
low molecular weight and other property. Thereby, the potent DPP-IVinhibitory fraction, P4-1, was need further separation and purification.
HPLC, the last purification step, usually provides the best purified
results [38]. Ji et al. found the purification folds of HPLC was as high as
43.973, which was much higher than that of ultrafiltration and gel
chromatography [13]. Fraction P4-1 was further purified by RP-HPLC.
As demonstrated in Fig. 3a, six fractions (P4-1–1 ~ P4-1–6) was sepa­
rated from P4-1. Fig. 3b shows the IC50 value corresponding to each
Fig. 3. RP-HPLC chromatogram of the DPPIV inhibitory activity fraction P4-1 on Sephadex G-15 gel chromatogram (a) and the IC50 value of each fraction (P4-1–1,
P4-1–2, P4-1–3, P4-1–4, P4-1–5 and P4-1–6) (b).
5
X. Xiang et al.
Journal of Chromatography B 1186 (2021) 122990
fraction. The IC50 value of P4-1–6 (0.59 ± 0.047 mg/mL) was signifi­
cantly lower than P4-1–1(2.09 ± 0.052 mg/mL), P4-1–2(1.67 ± 0.044
mg/mL), P4-1–3 (0.85 ± 0.041 mg/mL), P4-1–4(2.27 ± 0.09 mg/mL)
and P4-1–5(1.06 ± 0.058 mg/mL), meanwhile, peaks of P4-1–6
appeared successively bound by the fifth minute of elution time. This
may indicate that there were more than non-hydrophobic DPP-IV
inhibitory peptides. Song et al. reported that some DPP–IV inhibitory
peptides contain hydrophobic amino acids [39]. The fraction P4-1–6
exhibited the best DPP-IV inhibitory activity and was chosen to identify
peptide sequences.
Table 1
Peptides identified in collected fractions P4-1–6 and twenty-one peptides shown
in form were synthesized to evaluate their dipeptidyl peptidase-IV (DPP-IV)
inhibitory activity.
3.3. Identification of potential DPP-IV inhibitory peptides
The peptide sequences in fraction P4-1–6 were identified by LC-MS/
MS. The peptide sequences in fraction P4-1–6 were searched from
Uniprot-P. vannamei head by LC-MS/MS, and a total of 85 peptides were
identified, most of which were tripeptide and tetrapeptide (Supple­
mentary materials 1). Although the mechanism of action of DPP-IV
inhibitory peptides was not yet fully clear, some fixed structure fea­
tures in the peptides sequence could become critical factors in DPP-IV
inhibitory peptides. Many previous studies confirmed that a typical
characteristic of DPP-IV inhibitory peptides is alanine (A) or proline (P)
in the second position of the N-terminal. For instance, Nongonierma
et al. detected DPP-IV inhibitory peptides YPI from β-CN with the IC50
values of 35.0 ± 2.0 μM [40]. Bella et al. extracted peptides AP from the
brush border membrane of rat small intestinal mucosal cells with the
IC50 values of 2.43 mmol/L [41]. Gallego et al. found that the peptides
KA and AAATP derived from Spanish dry-cured ham showed the
stronger DPP-IV inhibitory activity and their IC50 values were 6.27 mM
and 6.47 mM respectively [24]. This phenomenon may attribute to the
similar structural features of these peptides to GLP-1, therefore, these
peptides replaced GLP-1 as the target of DPP-IV and reduced the effi­
ciency of DPP-IV. Besides, lots articles pointed out that some low mo­
lecular weight peptides were lack of alanine or proline but possessed
hydrophobic amino acid residues (I, W, L, V, and F) in the N-terminal
first position, which also were considered as effective inhibitors of DPPIV [42,43], since the hydrophobic amino acid of the peptides could
interact with the hydrophobic active site of the pocket of DPP-IV [44].
Jin et al. obtained DPP-IV inhibitory peptides VLATSGPG and LDKVFR
from Atlantic salmon skin with the IC50 values of 0.18 ± 0.02 mg/mL
(256.86 μM) and 0.10 ± 0.03 mg/mL (128.71 μM), correspondingly
[45]. Hong et al. extracted peptides WGDEHIPGSPYH from silver carp
swim bladder and its hydrolysate IPGSPY both possessed good inhibition
for soluble DPP-IV and promoted insulin secretion tested on INS-1 and
Caco-2 cells [46]. Among all peptides from P4-1–6, 21 peptides were
selected for further analysis, based on MS/MS spectrum of the peptides
score (>20), and their parent protein information was showed in Sup­
plementary Material 2. The mass spectrum matching scores of the
remaining peptides were too low to determine the reliability of the ex­
istence of these peptides, so no further analysis. The inhibitory activity
of those synthetic peptides was determined in vitro and the results were
showed as Table 1. Four novel DPP-IV inhibitory peptides (YPGE, VPW,
HPLY, YATP) had superior inhibitory capacity among them and their
MS/MS spectrum were shown in Fig. 4. Their IC50 value were 40.90 ±
2.76 μM, 174.781 ± 5.08 μM, 461.89 ± 3.23 μM, 475.33 ± 6.24 μM,
respectively, which were lower than many known food-derived peptide
IC50 value, such as IAAHFL (610.1 ± 82.6 L μM), EQLTKCEVFR (883 ±
36.8 μM), etc. As previously studies stated, most DPP-IV inhibitory
peptides were low molecular weight peptides, which was consistent with
our results [42]. The four peptides all could be degraded into Xaa-Pro or
Xaa-Pro-Yaa, which combined with the active sites of DPP-IV competi­
tively. In contrast with other three peptides, the tetrapeptide YPGE had
the strongest inhibition, but its DPP-IV inhibitory activity was not the
highest currently reported, this difference may need to be explained
from the mechanism. Interestingly, peptides FPR, VPW and FAGL all
possessed two typical structural features of DPP-IV inhibitory peptides,
Number
Sequence
Start-end
Mass (Da)
DPP-IV IC50 (μM)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Diprotin A
YANTP
YGGY
DRLY
EDR
VPW
YEY
YPGE
PLKD
HPLY
KDGQ
FPR
RLL
FGGF
YATP
WSL
GEGW
RFR
FAGL
YSH
VQPP
TLSK
IPI
f (1547–1551)
f (3492–3495)
f (3957–3960)
f (1293–1295)
f (4196–4198)
f (1360–1362)
f (2714–2717)
f (1339–1342)
f (597–600)
f (4249–4252)
f (1603–1605)
f (1365–1367)
f (1068–1071)
f (1564–1567)
f (4494–4496)
f (2179–2182)
f (1117–1119)
f (226–229)
f (2243–2245)
f (2289–2292)
f (736–739)
\
564.63
458.18
565.28
418.18
400.50
473.17
464.52
471.26
528.65
446.21
418.52
400.27
426.19
450.53
404.20
447.17
477.28
406.52
405.16
439.24
447.26
341.28
>1000
>1000
NO
NO
174.781 ± 5.08
>500
40.90 ± 2.76
>1000
461.89 ± 3.23
NO
>500
>1000
NO
475.33 ± 6.24
>500
>1000
NO
>500
>1000
>500
NO
3.97 ± 1.08
but their inhibitory activity was not the highest and was significantly
lower than YPGE. It seems to be slightly different from previous con­
clusions [47], which stated the DPP-IV inhibitory activity could be
enhanced due to the presence of a hydrophobic amino acid at the N- or
C-terminal position. We infer that this may be related to the more
complex structure of the peptide, which requires further exploration. Ji
et al. also used shrimp (Antarctic krill) as raw material to prepare DPPIV inhibitory peptides [13]. They obtained peptides AP and IPA with
IC50 values of 0.0530 mg/mL and 0.0370 mg/mL, which were higher
than the values of YPGE (0.019 mg/mL) and VPW (0.071 mg/mL) in this
article. In addition, we found that these sequences of the four peptides
have not yet appeared in other articles about DPP-IV inhibitory peptides
by querying BIOPEP (http://www.uwm.edu.pl/biochemia/index.php/
en/biopep). The results indicated that P. vannamei head has potential
for the release of DPP-IV inhibitory peptides.
3.4. Molecular docking simulations of DPP-IV inhibitory peptides to DPPIV
Molecular docking study is always used to explore the interaction
mechanism between the ligand and receptor. The molecular docking of
the DPP-IV monomer and the selected four peptides was simulated by
AutoDock Vina software, which outputs the result in the form of Affinity
that is an important indicator of whether the ligand can bind to the
receptor molecule effectively. The affinity score is mainly based on a
comprehensive calculation of the spatial effects, repulsion, hydrogen
bonding, hydrophobic interactions, as well as molecular flexibility
equivalence of the receptor-ligand complex. A lower docking score
represents a better molecular-binding conformation between peptides
and DPP-IV [48]. In this article, the peptide YPGE depicted the lowest
docking score (− 8.3 kcal/mol) than HPLY (− 7.9 kcal/mol), VPW (− 8.2
kcal/mol), YATP (− 7.5 kcal/mol), and this is consistent with the results
of in vitro experiments. This may be due to the presence of proline which
could highly enhance the interaction between DPP-IV inhibitors and
DPP-IV [5]. Though these four docking scores were higher than Diprotin
A (− 9.5 kcal/mol), it cannot hide that these four peptides have formed
stable complexes with DPP-IV. In order to observe docking way more
accurately, the 3D diagrams of the combination of the four peptides and
DPP-IV are shown in Fig. 5 (a-d). In Fig. 5(a), six hydrogen bonds
(Glu205, Ser209, Tyr547, Arg358, Glu361, Ser360) were formed
6
X. Xiang et al.
Journal of Chromatography B 1186 (2021) 122990
Fig. 4. MS/MS spectrum of four superior DPP-IV inhibitory peptide from the purified P4-1–6 fraction. YPGE (a), HPLY (b), YATP (c), VPW (d).
7
X. Xiang et al.
Journal of Chromatography B 1186 (2021) 122990
Fig. 5. 3D diagrams of the combination of the four superior peptides and DPP-IV. (a): YPGE-DPP-IV; (b): HPLY-ADPP-IV; (a): YATP-DPP-IV; (a): VPW-DPP-IV.
between the peptide YPGE and DPP-IV residues, in addition, amino acid
residues Ile374, Ile405, Glu361, Pro359, Gly355, Arg356, Phe357,
Glu206, Tyr666 formed hydrophobic interactions with PYGE. Ser360
and Glu209 belong to major residues in active sites S1 and S2, respec­
tively. Tyr547, Phe357 and Arg358 existed in the cavity of S3 [49].
Compared with the previous conclusion, DPP-IV enzyme activity also
Fig. 6. Stability the four synthetic DPP-IV inhibitory peptides. (a) Simulated gastrointestinal stability with gastric phase (pH 2.0, 120 min) and intestinal phase (pH
7.5, 120 min). (b) Thermal stability at different temperature (37, 50, 60, 70, 80, 90, 100 or 121 ◦C) for 30 min; (c) pH stability at pH 1.0, 2.0, 4.0, 6.0, 8.0, 10.0
or 11.0.
8
X. Xiang et al.
Journal of Chromatography B 1186 (2021) 122990
could be inhibited by hydrogen bonding and hydrophobic interaction
between DPP-IV and inhibitory peptides. DPP-IV possesses two active
pockets and one connection area which relates to inhibitor; the S1
pocket including residues His740, Asn710 and Ser630; the S2 area
including Glu206, Arg125 and Glu205, and the S3 pocket consists of
Tyr547, Arg358, and Phe357 [5]. The docking of the remaining three
peptides shown in Fig. 4(b-d), all of the peptides had interactions with
the S1, S2, or S3 pocket residues, but there was different docking
numbers and sites or areas, which is perhaps one of the reasons for the
diversity between them. It is worth noting that, compared with Diprotin
A, there is no double salt bridge formed between four peptides and DPPIV, so their inhibitory activity was weaker than Diprotin A in vitro. As
well, there was hydrogen bond formed by the medium polar electro­
philic group in the peptide due to the carboxyl terminus stabilized two
ion pairs with Asn710 and Try547, which was also another reason why
the inhibitory activity of these four peptides was inferior to Diprotin A
[50]. Currently, it is common to use molecular docking to explore the
inhibition mechanism of DPP-IV, however, there are still many unclear
aspects in the mechanism. The investigation of the mechanism still
needs to be strengthened.
the adjusted pH might affect bioactive activity of peptides by changing
the charges in peptides [52]. Therefore, the four DPP-IV inhibitory
peptides activity exhibited good stability at pH 1.0–10.0, suggesting that
it may be incorporated into food systems with pH range at 1.0–10.0
while maintaining the DPP-IV inhibitory activity.
4. Conclusions
In summary, P. vannamei head was first reported to prepare DPP-IV
inhibitory peptides. P. vannamei head animal protease hydrolysate
showed high DPP-IV inhibitory activity in vitro. Ultrafiltration, gel
chromatography, and RP-HPLC purification increased the DPP-IV
inhibitory activity of peptide fractions compared with the initial hy­
drolysate. The strongest fraction was identified by LC-MS/MS and four
peptides showed superior inhibition. The novel DPP-IV inhibitory pep­
tide YPGE illustrated the highest DPP-IV inhibitory activity. Molecular
docking effectively explored their mechanism of action through com­
bined hydrogen bonds and hydrophobic regions. Stability tests sug­
gested that these four peptides could maintain good stability during food
processing and gastrointestinal digestion. The results demonstrated that
P. vannamei head can be considered as a potential natural raw of DPP-IV
inhibitor. However, this article was limited by in vitro analysis and lack
of in vivo testing. Hence, further research in vivo is necessary to validate
whether the DPP-IV inhibitory peptides could be used as a dietary
supplement against type 2 diabetes.
3.5. Stability of DPP-IV inhibitory peptides activity
The stability of bioactive peptides activity has received growing in­
terest with the importance of bioactive peptides gradually emerging
[51]. The gastrointestinal stability of the four synthetic DPP-IV inhibi­
tory peptides activity was illustrated in Fig. 6a. The decrease of relative
DPP-IV inhibitory activity of the four peptides was limited throughout
digestion simulation process, where the maximum was less than 6%.
Interestingly, the relative DPP-IV inhibitory activity of peptides VPW
and YATP increased by 7.07% and 11.81%, respectively, within 30 min
and 60 min during in vitro gastric digestion. In addition, the DPP-IV
inhibitory activity of the peptide YATP has been increased by 12.44%
in vitro intestinal digestion. The phenomenon was similar to previous
article, which found that the DPP-IV inhibitory activity of peptides
PGVGGPLGPIGPCYE and CAYQWQRPVDRIR increased by 8–12% after
gastrointestinal digestion simulation [23]. We speculate that the pep­
tides were degraded by gastrointestinal proteases into Xaa-Pro or XaaPro-Yaa, which brought about an increase in DPP-IV inhibitory activ­
ity due to the release of more potent DPP-IV inhibitory peptides [5].
Whether this structure would be further degraded by digestive proteases
was the reason for their subsequent changes in DPP-IV inhibitory ac­
tivity. It indicated that the four peptides maintained their DPP-IV
inhibitory activity well during in vitro gastrointestinal digestion.
The impacts upon DPP-IV inhibitory activity of the four peptides at
different temperatures were showed in Fig. 6b. As the temperature
varied from 25 ◦ C to 121 ◦ C, the DPP-IV inhibitory activity of the four
peptides had different change trend, and the decrease was the most
remarkable after being treated at 121 ◦ C for 30 min. However, the
relative DPP-IV inhibitory activity was still higher than 90%. It was
noted that peptides treated at the same temperature had different
changes in bioactive peptide activity, which was mainly caused by the
type of peptides, the size of peptides and the proportion of hydrophobic
domain in peptides [52]. The test indicated that the four peptides
maintained preferable thermal stability and can thus be subjected to
thermal treatment during food processing. The effect of pH values
ranged from 1.0 to 11.0 on the DPP-IV inhibitory activity of the four
peptides was depicted in Fig. 6c. In the pH varied from 1.0 to 10.0, the
slight fluctuations of DPP-IV inhibitory activity were observed. At the
strong alkaline conditions (pH > 10.0), there was an important reduc­
tion of DPP-IV inhibitory activity in the four peptides, which was similar
to previous studies. Wu et al. reported that the bioactivity peptides
derived from bovine casein noticeably decreased under extremely
alkaline conditions. A possible reason could be that some bioactive
peptides were further degraded into inactive fragments under strong
acidic or alkaline [53]. Meanwhile, Kittiphattanabawon et al. found that
CRediT authorship contribution statement
Xi Xiang: Formal analysis, Methodology, Data curation, Writing –
original draft. Meng Lang: Investigation, Data curation, Methodology.
Yan Li: Investigation, Methodology, Data curation, Validation, Funding
acquisition. Xia Zhao: Investigation, Methodology. Huimin Sun:
Investigation, Methodology. Weiwei Jiang: Investigation, Methodol­
ogy. Ling Ni: Investigation, Methodology. Yishan Song: Data curation,
Funding acquisition, Conceptualization, Supervision, Writing – review
& editing.
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.
Acknowledgment
This work was supported by the National Key Research and Devel­
opment Program of China (2019YFD0902000).
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jchromb.2021.122990.
References
[1] X. Yuan, X. Gu, J. Tang, Purification and characterisation of a hypoglycemic
peptide from Momordica Charantia L. Var. abbreviata Ser, Food Chem. 111 (2008)
415–420.
[2] B. Salehi, A. Ata, N, V. A. K., et al., Antidiabetic potential of medicinal plants and
their active components. Biomolecules 9 (2019) 551–563.
[3] E.J. Benjamin, P.S. Chen, D.E. Bild, et al., Prevention of atrial fibrillation: report
from a national heart, lung, and blood institute workshop, Circulation 119 (2009)
606–618.
[4] X. Wang, M. Han, M. Zhang, Y. Wang, Y. Ren, T. Yue, Z. Gao, In vitro evaluation of
the hypoglycemic properties of lactic acid bacteria and its fermentation
adaptability in apple juice, Lwt 136 (2021) 110363, https://doi.org/10.1016/j.
lwt.2020.110363.
[5] F. Xu, Y. Yao, X. Xu, et al., Identification and quantification of DPP-IV-inhibitory
peptides from hydrolyzed-rapeseed-protein-derived napin with analysis of the
9
X. Xiang et al.
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
Journal of Chromatography B 1186 (2021) 122990
interactions between key residues and protein domains, J. Agric. Food Chem. 67
(2019) 3679–3690.
E. Valencia-Mejia, K.A. Batista, J.J.A. Fernandez, et al., Antihyperglycemic and
hypoglycemic activity of naturally occurring peptides and protein hydrolysates
from easy-to-cook and hard-to-cook beans (Phaseolus vulgaris L.), Food Res. Int.
121 (2019) 238–246.
K. Imamura, Y. Tsuyama, T. Hirata, Identification and characterization of a novel
fermented substance produced by edible Aspergillus oryzae AO-1 that inhibits DPPIV activity, J. Biosci. Bioeng. 111 (2011) 37–40.
B.N. Conway, D.M. Long, M.K. Figaro, et al., Glycemic control and fracture risk in
elderly patients with diabetes, Diabetes Res. Clin. Pract. 115 (2016) 47–53.
F. Rivero-Pino, F.J. Espejo-Carpio, E.M. Guadix, Production and identification of
dipeptidyl peptidase IV (DPP-IV) inhibitory peptides from discarded Sardine
pilchardus protein, Food Chem. 328 (2020) 127096, https://doi.org/10.1016/j.
foodchem.2020.127096.
H. Fitipaldi, M.I. McCarthy, J.C. Florez, et al., A global overview of precision
medicine in type 2 diabetes, Diabetes 67 (2018) 1911–1922.
A. Lemes, L. Sala, J. Ores, A. Braga, M. Egea, Kátia Fernandes, A review of the latest
advances in encrypted bioactive peptides from protein-rich waste, Int. J. Mol. Sci.
17 (6) (2016) 950, https://doi.org/10.3390/ijms17060950.
Meester, L. A.-M. D. C. S. S. D., Dipeptidyl-peptidase IV from bench to bedside An
update on structural properties, functions, and clinical aspects of the enzyme DPP
IV, Crit. Rev. Clin. Lab. Sci. 40 (2003) 209–294.
W. Ji, C. Zhang, H. Ji, Purification, identification and molecular mechanism of two
dipeptidyl peptidase IV (DPP-IV) inhibitory peptides from Antarctic krill
(Euphausia superba) protein hydrolysate, J. Chromatogr. B Analyt. Technol.
Biomed. Life Sci. 1064 (2017) 56–61.
A.K. Singh, Dipeptidyl peptidase-4 inhibitors: novel mechanism of actions, Indian
J. Endocrinol. Metab. 18 (2014) 753–759.
R. Mentlein, G. B. a. S. W. E., Dipeptidyl-peptidase IV hydrolyses gastric inhibitory
polypeptide,glucagon-like peptide-l(7-36)amide, peptide histidine methionine and
is responsible for their degradation in human serum, Eur. J. Biochem. 214 (1993)
829–835.
S. Chatterjee, K. Khunti, M.J. Davies, Type 2 diabetes, The Lancet 389 (2017)
2239–2251.
S.A. Ross, E.A. Gulve, M. Wang, Chemistry and biochemistry of type 2 diabetes,
Chem. Rev. 104 (3) (2004) 1255–1282.
A.M. Bower, L.M. Real Hernandez, M.A. Berhow, E.G. de Mejia, Bioactive
compounds from culinary herbs inhibit a molecular target for type 2 diabetes
management, dipeptidyl peptidase IV, J. Agric. Food Chem. 62 (26) (2014)
6147–6158.
P. Aschner, M.S. Kipnes, J.K. Lunceford, et al., Effect of the dipeptidyl peptidase-4
inhibitor sitagliptin as monotherapy on glycemic control in patients with type 2
diabetes, Diabetes Care 29 (2006) 2632–2637.
S. Sun, G. Zhang, H. Mu, et al., The mixture of corn and wheat peptide prevent
diabetes in NOD mice, J. Funct. Foods 56 (2019) 163–170.
T. Hatanaka, Y. Inoue, J. Arima, et al., Production of dipeptidyl peptidase IV
inhibitory peptides from defatted rice bran, Food Chem. 134 (2012) 797–802.
B. Konrad, D. Anna, S. Marek, et al., The evaluation of dipeptidyl peptidase (DPP)IV, alpha-glucosidase and angiotensin converting enzyme (ACE) inhibitory
activities of whey proteins hydrolyzed with serine protease isolated from asian
pumpkin (cucurbita ficifolia), Int. J. Pept. Res. Ther. 20 (2014) 483–491.
S.L. Huang, C.L. Jao, K.P. Ho, et al., 3 Dipeptidyl-peptidase IV inhibitory activity of
peptides derived from tuna cooking juice hydrolysates, Peptides 35 (2012)
114–121.
M. Gallego, M.C. Aristoy, F. Toldra, Dipeptidyl peptidase IV inhibitory peptides
generated in Spanish dry-cured ham, Meat Sci. 96 (2014) 757–761.
A. Casanova-Marti, F.I. Bravo, J. Serrano, et al., Antihyperglycemic effect of a
chicken feet hydrolysate via the incretin system: DPP-IV-inhibitory activity and
GLP-1 release stimulation, Food Funct. 10 (2019) 4062–4070.
A.B. Nongonierma, S. Paolella, P. Mudgil, et al., Dipeptidyl peptidase IV (DPP-IV)
inhibitory properties of camel milk protein hydrolysates generated with trypsin,
J. Funct. Foods 34 (2017) 49–58.
A. Kanekanian, Bioactive proteins and peptides as functional foods and
nutraceuticals, Int. J. Dairy Technol. 64 (2011) 316–347.
R. Madrigal, U.D.T. da Silva, C.P.D. Tavares, et al., Use of native and non-native
shrimp (Penaeidae, Dendrobranchiata) in world shrimp farming, Rev. Aquaculture
10 (2018) 899–912.
S. Ren, P.B. Mather, B. Tang, et al., Levels of genetic diversity and inferred origins
of Penaeus vannamei culture resources in China: Implications for the production of
a broad synthetic base population for genetic improvement, Aquaculture 491
(2018) 221–231.
T.H. Tinh, S. Elayaraja, M. Mabrok, et al., Antibacterial spectrum of synthetic
herbal-based polyphenols against Vibrio parahaemolyticus isolated from diseased
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
10
Pacific whiteleg shrimp (Penaeus vannamei) in Thailand, Aquaculture 533 (2021)
736070–736091.
J. Sivakamavalli, R. Arthur James, K. Park, et al., Purification of WAP domaincontaining antimicrobial peptides from green tiger shrimp Peaneaus semisulcatus,
Microb. Pathog. 140 (2020) 103920–103937.
I. Joshi, J. K, K. Noorani, P. M., et al., Isolation and characterization of angiotensin
I-converting enzyme (ACE-I) inhibition and antioxidant peptide from by-catch
shrimp (Oratosquilla woodmasoni) waste, Biocatal. Agric. Biotechnol. 29 (2020)
101770–101787.
C. Zhang, Y. Zhang, Z. Wang, et al., Production and identification of antioxidant
and angiotensin-converting enzyme inhibition and dipeptidyl peptidase IV
inhibitory peptides from bighead carp (Hypophthalmichthys nobilis) muscle
hydrolysate, J. Funct. Foods 35 (2017) 224–235.
P.M. Nielsen, D. Petersen, Dambmann, Improved method for determining food
protein degree of hydrolysis, J. Food Sci. Technol. 66 (2001) 642–646.
X. Kong, L. Zhang, W. Song, et al., Separation, identification and molecular binding
mechanism of dipeptidyl peptidase IV inhibitory peptides derived from walnut
(Juglans regia L.) protein, Food Chem. 347 (2021) 1290–1305.
P.A. Harnedy-Rothwell, C.M. McLaughlin, M.B. O’Keeffe, et al., Identification and
characterisation of peptides from a boarfish (Capros aper) protein hydrolysate
displaying in vitro dipeptidyl peptidase-IV (DPP-IV) inhibitory and insulinotropic
activity, Food Res. Int. 131 (2020) 108989–108997.
A.B. Nongonierma, S. Paolella, P. Mudgil, et al., Identification of novel dipeptidyl
peptidase IV (DPP-IV) inhibitory peptides in camel milk protein hydrolysates, Food
Chem. 244 (2018) 340–348.
A.B. Nongonierma, R.J. FitzGerald, Susceptibility of milk protein-derived peptides
to dipeptidyl peptidase IV (DPP-IV) hydrolysis, Food Chem. 145 (2014) 845–852.
J.J. Song, Q. Wang, M. Du, et al., Identification of dipeptidyl peptidase-IV
inhibitory peptides from mare whey protein hydrolysates, J. Dairy Sci. 100 (2017)
6885–6894.
A.B. Nongonierma, C. Cadamuro, A. Le Gouic, et al., Dipeptidyl peptidase IV (DPPIV) inhibitory properties of a camel whey protein enriched hydrolysate
preparation, Food Chem. 279 (2019) 70–79.
A.M. Bella, R.H. Erickson, Y.S. Kim, Rat intestinal brush border membrane
dipeptidyl-aminopeptidase IV Kinetic properties and substrate specificities of the
purified enzyme, Arch. Biochem. Biophys. 218 (1) (1982) 156–162.
A.B. Nongonierma, R.J. FitzGerald, Features of dipeptidyl peptidase IV (DPP-IV)
inhibitory peptides from dietary proteins, J. Food Biochem. 43 (1) (2019) e12451,
https://doi.org/10.1111/jfbc.12451.
A.B. Nongonierma, R.J. FitzGerald, An in silico model to predict the potential of
dietary proteins as sources of dipeptidyl peptidase IV (DPP-IV) inhibitory peptides,
Food Chem. 165 (2014) 489–498.
A.B. Nongonierma, C. Mazzocchi, S. Paolella, et al., Release of dipeptidyl peptidase
IV (DPP-IV) inhibitory peptides from milk protein isolate (MPI) during enzymatic
hydrolysis, Food Res. Int. 94 (2017) 79–89.
R. Jin, X. Teng, J. Shang, D. Wang, N. Liu, Identification of novel DPP-IV inhibitory
peptides from Atlantic salmon (Salmo salar) skin, Food Res. Int. 133 (2020)
109161, https://doi.org/10.1016/j.foodres.2020.109161.
H. Hong, Y. Zheng, S. Song, et al., Identification and characterization of DPP-IV
inhibitory peptides from silver carp swim bladder hydrolysates, Food Biosci. 38
(2020) 100748–100758.
C.L. Jia, N. Hussain, O. Joy Ujiroghene, et al., Generation and characterization of
dipeptidyl peptidase-IV inhibitory peptides from trypsin-hydrolyzed alphalactalbumin-rich whey proteins, Food Chem. 318 (2020) 126333–126342.
D.H. Utomo, N. Widodo, M. Rifa’i, Identifications small molecules inhibitor of p53mortalin complex for cancer drug using virtual screening, Bioinformation 8 (9)
(2012) 426–429.
S.Q. Pantaleao, E.A. Philot, P.T. de Resende-Lara, et al., Structural dynamics of
DPP-4 and its influence on the projection of bioactive ligands, Molecules 23 (2018)
490–503.
Velazquez, C. G.-A. M.-C. B. I. D. M. A. S.-F. J. B. M.-J. C. S., Design and discovery
of a novel dipeptidyl-peptidase IV (CD26)-based prodrug approach, J. Med. Chem.
49 (2006) 5339–5351.
S. Ketnawa, S. Benjakul, O. Martinez-Alvarez, et al., Fish skin gelatin hydrolysates
produced by visceral peptidase and bovine trypsin: bioactivity and stability, Food
Chem. 215 (2017) 383–390.
P. Kittiphattanabawon, S. Benjakul, W. Visessanguan, et al., Gelatin hydrolysate
from blacktip shark skin prepared using papaya latex enzyme: antioxidant activity
and its potential in model systems, Food Chem. 135 (2012) 1118–1126.
Y. Fu, J.F. Young, T.K. Dalsgaard, M. Therkildsen, Separation of angiotensin Iconverting enzyme inhibitory peptides from bovine connective tissue and their
stability towards temperature, pH and digestive enzymes, Int. J. Food Sci. Technol.
50 (5) (2015) 1234–1243.
Descargar