International Journal of Biological Macromolecules 169 (2021) 384–395

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Effective separation of prolyl endopeptidase from Aspergillus Niger by

aqueous two phase system and its characterization and application
Bin Jiang a, Meichan Wang a, Xiaojing Wang a, Shuang Wu b, Dongmei Li a, Chunhong Liu a,
Zhibiao Feng a,⁎, Jie Li c,⁎
a
Department of Applied Chemistry, Northeast Agricultural University, Harbin, Heilongjiang 150030, People's Republic of China
b
Heilongjiang Eco-meteorology Center, Harbin, Heilongjiang 150030, People's Republic of China
c
College of Life Science, Northeast Agricultural University, Harbin, Heilongjiang 150030, People's Republic of China

a r t i c l e i n f o a b s t r a c t

Article history: Aspergillus niger prolyl endopeptidase (An-PEP) has become a research focus because of its advantages in specif-
Received 5 October 2020 ically cleaving the C-terminal peptide bond of proline residues, especially it was an industrial food-grade acidic
Received in revised form 27 November 2020 PEP. Aqueous two-phase system (ATPS) was first applied for separating An-PEP from fermentation broth. Via re-
Accepted 15 December 2020
sponse surface method (RSM) experiment, an effectively separation of An-PEP was achieved by ATPS contain-
Available online 19 December 2020
ing27% (w/w) ethanol and 14.5% (w/w) (NH4)2SO4 at pH 6.0 with the recovery of 90.29 ± 0.23% and
Keywords:
purification coefficient of 15.35 ± 0.30. The purified An-PEP was characterized by sodium dodecyl sulfate poly-
Aqueous two-phase system acrylamide gel electrophoresis (SDS–PAGE), fourier transform infrared (FTIR) and fluorescence spectrometry.
Prolyl endopeptidase The optimum temperature and pH of An-PEP were 40 °C and 4.5–5.0, respectively. An-PEP was activated and sta-
Aspergillus niger bilized by Ca2+ but inhibited by Fe3+. The enzymatic application of purified An-PEP was evaluated by hydrolyz-
Purification ing egg white protein (EWP) to prepare bioactive peptides. The obtained hydrolysates had good scavenging
Properties ability of OH• and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radicals, angiotensin
Bioactive peptides converting enzyme (ACE) inhibitory activity and anti-gout activity. This research realized a low-cost, high-
efficiency and simple separation technology of An-PEP and provided a broader idea for the preparation of bioac-
tive peptides and the application of An-PEP.
© 2020 Elsevier B.V. All rights reserved.

1. Introduction
Prolyl endopeptidase (PEP, EC 3.4.21.26) was first discovered by
Walter as an oxytocin-degrading enzyme [1]. PEP was also known as
prolyl oligopeptidase or post-proline cleaving enzyme [2], which
existed in a number of plants, mammals, bacteria and fungi [3,4]. PEPs
were a unique enzymes that had the possibility of preferentially cleaved
peptides at the carboxyl side of proline residues, followed by Leucine,
Methionine, Phenylalanine, and Alanine residues. As previously re-
ported, PEPs only cleaved peptide substrates and hydrolyzed peptide
fragments of less than 30 amino acids [5]. However, PEP from Aspergillus
niger, different from PEP from other microorganisms or mammalian,
had the ability to cleave macromolecular protein, which did not require
any other pre-hydrolysis steps with other broad specificity proteinase
activity [6]. Moreover, An-PEP was different with the PEPs which were
derived from some pathogenic bacteria, as Pseudomonas sp. KU-22 [1]
and Stenotrophomonas maltophilia [5], it was industrial food-grade PEP
⁎ Corresponding author at: No. 600, Changjiang Road, Xiangfang District, Harbin City,
Heilongjiang Province, People's Republic of China.
E-mail addresses: fengzhibiao@neau.edu.cn (Z. Feng), lijie_neau@126.com (J. Li).
with an optimal acid pH levels. Previous studies had proved that, in
the industry, An-PEP could be used to debitter protein hydrolyzates
[7], prevent chill-haze in beer and eliminate gluten by degradation of
proline-rich proteins [8,9], and alleviate celiac disease symptoms [10].
Therefore, a deeper study of the An-PEP was the focus of this paper. In
addition, as known from previous studies, there were no reports on
the separation of An-PEP using ATPS.
The separation and purification of enzymes was the premise of
studying the properties, structure and functions of enzymes. The pur-
pose of enzyme purification was to separate and purify enzymes from
the organism with high purity and good yield. From previous research,
PEP had been separated and purified by several methods, including salt-
ing precipitation [11], dialysis [7], gel filtration chromatography [1], ion
exchange chromatography [3] and affinity chromatography [12]. Gener-
ally, the purity and yield of enzymes gained by a single purification
method were unsatisfactory. The application of traditional single extrac-
tion method involves the disadvantages of low purification efficiency.
On the other hand, when dealing with samples with uneven distribu-
tion of particle size and high viscosity, a single purification method
may be challenging. So combination of multiple methods was generally
used to separate and purify enzymes. Wadhawan et al. [11] reported

https://doi.org/10.1016/j.ijbiomac.2020.12.120
0141-8130/© 2020 Elsevier B.V. All rights reserved.
B. Jiang, M. Wang, X. Wang et al.
that ammonium sulphate precipitation, DEAE sepharose column and
phenyl sepharose CL-4B hydrophobic interaction column were com-
bined to separate and purify PEP from Setaria cervi. In addition, Edens
et al. [7] combined bacitracin column chromatography, dialysis, ultrafil-
tration with agitated cell PM-10 membrane and Superdex 75 column to
obtain highly purified prolyl endoprotease from Aspergillus niger. How-
ever, the combination of multiple methods suffered the disadvantages
of batch operation, low process integration ability, laborious processing
cycles, high energy input and high cost. In particular, a certain amount
of target compound was lost in each processing stage. So, it was signif-
icant to study a simple and economical neomethod for separating An-
PEP from fermentation broth.
ATPS was generally composed of two kinds of immiscibility poly-
mers or one polymer and salts, and had been effectively applied for
the purification, separation, extraction and enrichment of proteins, pro-
teases and some other biomolecules [13,14]. Tremendous efforts have
been done to research various types and applications of ATPSs in sepa-
ration of various biomaterials [15,16]. However, because of the exorbi-
tant cost of the polymer and the complexity of separating extracted
molecules during the back extraction processes, industrial upscaling of
these ATPSs still remained compromised [17]. While, small molecular
alcohol/salt systems were considered to have the more advantages
than other ATPS systems of fast phase separation, low viscosity, gently
environment, recyclable [18].The advantage of easily recovered by
evaporation and crystallization made it easy to separate the target com-
pounds from the alcohol-rich phase, thus reducing the cost of purifica-
tion and enrichment and simplifying the later process of industrial
application and production [19]. Due to the above advantages of small
molecular alcohol/salt system, the research on it has become a hot
topic. In addition, the advantages of sulfates in promoting hydrophobic
interactions between proteins and preferentially being removed from
the protein surface, as well as the traditional method of extracting pro-
teins by ammonium sulfate precipitation, (NH4)2SO4 was preferred as a
component of ATPS. Therefore, the separation and purification of An-
PEP with alcohol/(NH4)2SO4 ATPS was an additional focus of this re-
search paper.
EWP was an important functional and nutritional production in eggs
and one of the significant sources of bioactive peptides. However, aller-
gens in EWP, such as ovalbumin, ovotransferrin, lysozyme and egg nu-
cleus, would cause harm to sensitive people [20]. Therefore, how to
reduce the allergenicity of eggs had attracted widespread attention. Re-
searchers had found that enzymatic hydrolysis could reduce the allerge-
nicity of EWP, and the enzymatic hydrolysis method could prepare
polypeptides without any chemical or physical induction treatment,
and the nutritional and functional values of the hydrolysates could be
well preserved. Therefore, it was envisaged to prepare polypeptides by
enzymatic hydrolysis while reducing the allergenicity of EWP. In addi-
tion, many studies had shown that the EWP hydrolysates or peptides
hydrolyzed by one or more enzymes, such as alkaline protease, trypsin,
pepsin, papain and flavor enzymes [21,22], had bioactivity and could re-
duce the allergenicity of EWP [23–25]. And these studies also showed
that the antioxidant activity of these peptides was higher than that of
the proteins, which might be caused by the destruction of the tertiary
structure of the protein by enzymatic hydrolysis. From all the above,
they provided an important theoretical basis for EWP peptides as
health food.
Owing to An-PEP was a research focus in food and medicine [7–10],
and there was no ATPS used to separate it. Therefore, with the purpose
of developing a new, high efficiency, low-cost and simple separation
methodology, ATPS was firstly used for separation and purification of
An-PEP from Aspergillus niger fermentation broth. In order to achieve
high activity ratio and industrialization, (NH4)2SO4 and ethanol were
chosen as the components of ATPS to separate An-PEP. The RSM exper-
iment was carried out on the basis of a single-factor experiment to get
the optimum separation efficiency. Then SDS–PAGE, FTIR and fluores-
cence spectrometry were used to characterize the purified An-PEP.
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International Journal of Biological Macromolecules 169 (2021) 384–395
The physical and chemical properties of An-PEP were studied, such as
the molecular weight, the optimum temperature and pH, and the influ-
ence of metal ions, etc. Furthermore, An-PEP was applied to hydrolyze
EWP to produce bioactive peptides, and the OH• and ABTS free radicals
scavenging activity, ACE inhibitory activity and anti-gout activity of
EWP hydrolysates were evaluated.
2. Materials and methods
2.1. Instruments
The T6 UV–Visible spectrophotometer was got from Beijing Purkinje
General Instrument Co., Ltd.. The Spark10M multi-function microplate
reader was received from Tecan Co., Ltd. (Teken, Swiss). The Biorad
Mini-PROTEAN Tetra Cells 4-Gel 165–8004 was obtained from Bio-Rad
Co., Ltd. (Guangzhou China). Other instruments were shown in
Supplemental materials (S1).
2.2. Reagents
Aspergillus niger fermentation broth was supplied by College of Life
Sciences of Northeast Agricultural University. Substrate (Ala-Ala-Pro-
pNA) was synthesized by Sangon Biotech (Shanghai). Ethanol was pur-
chased from Tianjin Fuyu Fine Chemical Co., Ltd. (Tianjin, China).
Disodium hydrogen phosphate (Na2HPO4), monometallic sodium or-
thophosphate (NaH2PO4) and ammonium sulfate ((NH4)2SO4) were re-
ceived from Beijing Yili Fine Chemicals Co., Ltd. (Beijing, China). All
reagents have not been further purified. Other reagents were shown
in Supplemental materials (S1).
2.3. Preparation of feedstock
Specifically, the source of An-PEP was different from previous stud-
ies. Using gene replacement technology, the prolyl endopeptidase gene
prot A in Aspergillus niger TH2 replaced the glucoamylase gene gla A and
knocked out the acid-stable α-amylase gene asa A, and ammonium sul-
fate was added into the fermentation medium. The above operations
eliminated three main secreted background proteins. Finally, the high
yield and purity prolyl endopeptidase was obtained. The fermentation
medium (1 L) components of Aspergillus niger mainly included 100 g
soybean meal powder, 20 g corn syrup and 20 mL glucose. The fermen-
tation broth of Aspergillus niger was subjected to simple separation to
get a crude extract. And the crude extract was placed in a 4 °C freezer
for use.
2.4. ATPS preparation
The ATPSs were made by adding an appropriate weight of ammo-
nium sulfate stock solution and ethanol and the crude extract (1 g)
into 10 mL graduated centrifuge tubes, then, the pH of the mixtures
was adjusted with 1 mol/L NaOH and the final weight of the system
was adjusted to 10 g with ultrapure water. The compounds were
mixed fully on a rotary mixer for 2 min at room temperature and centri-
fuged at 2000 rpm for 20 min. Then the top and bottom phase volumes
were measured, respectively. And the top and bottom phase samples
were collected to measure An-PEP activity and protein concentration.
The top and bottom phases of ATPS were dialyzed against distilled
water in a 1.4 K Da dialysis bag for 24 h to remove ethanol and ammo-
nium sulfate, and then concentrated to obtain ATPS top and bottom
phase concentrate, respectively. The bottom concentrates (An-PEP con-
centrates) were stored at −20 °C for use. And the concentrates were ly-
ophilized to obtain An-PEP lyophilized powder.
B. Jiang, M. Wang, X. Wang et al. International Journal of Biological Macromolecules 169 (2021) 384–395

2.5. Protein determination

Y ¼ A0 þ Ai ∑Xi þ Aij ∑Xi Xj þ Aij ∑X2j ði≠jÞ ð5Þ
Protein contents of top and bottom phases of ATPS were measured
according to Shashidhara [26]. Different concentrations of albumin where Y is the response, Xi and Xj represent the factors, A0, Ai, Aii and Aij
from bovine serum were used as a standard to make a standard curve. are regression coefficient of the intercept, linear, quadratic and interac-
1 mL of sample and 5 mL of Coomassie Brilliant Blue G-250 reagent tion coefficients, respectively. i and j range from 1 to 3. The statistical
were mixed thoroughly and incubated at room temperature for 5 min. significance evaluation method of model was F-test.
Then the absorbance at 595 nm was recorded against the reagent
blank. In order to prevent the interference from the phase composition 2.9. Electrophoresis anslysis
in the top phase, samples containing the same phase composition with-
out protein were analyzed as blanks. The crude extracts, ATPS top phase concentrate and ATPS bottom
phase concentrates, were diluted with ultrapure water to the same pro-
2.6. An-PEP activity determination tein concentration. SDS-PAGE was used to evaluate the distribution of
protein in the top and bottom phases of ATPS and the purity of An-
An-PEP activity was determined using the method reported by Jiang PEP by 8% bis-acrylamide homogeneous gel. The gel was stacked by run-
[27]. The substrate (Ala-Ala-Pro-pNA) was dissolved in 1, 4-dioxane so- ning it for about 20 min at a constant voltage of 80 V and separated by
lution (40%, v/v) to prepare a 10 mM solution. 80 μL of citrate/disodium running it for 55 min at a constant voltage of 120 V [28]. After electro-
phosphate buffer (0.1 mol/L, pH 5.0), 10 μL of An-PEP solution and sub- phoresis, the gel was dyed for 30 min with Coomassie brilliant blue R-
strate solution were mixed and incubated for 10 min at 40 °C. The absor- 250 and decolorized with decolorizing solution.
bance of reaction products was monitored at 410 nm.
2.10. Characterization of physicochemical properties of An-PEP
2.7. Definition of parameters in ATPSs
2.10.1. FTIR anslysis
Purification factor (PF), partition coefficient of An-PEP activity (Ke), The concentrates were heated in a water bath at various tempera-
and An-PEP recovery in the bottom phase (Y) were used to evaluate tures (50, 60, 70, 80 and 90 °C) for 30 min, and lyophilized to obtain
the separation and purification of An-PEP and were calculated by the An-PEP lyophilized powder specially used for fourier transform infrared
following formulas: spectroscopy analysis. The FTIR was measured based on the method re-
ported by Zhang [29] after slightly modified. 2 mg of An-PEP lyophilized
Eb
Ke ¼ ð1Þ powder was mixed together with 200 mg of KBr, the mixture was baked
Et for 10 min by an infrared ray roast lamp and pressed into a mold. It was
measured by scanning 32 scans with a resolution of 4 cm−1 in the range
RK e
Y¼  100% ð2Þ of 400–4000 cm−1. Infrared data fitting was made by OPUS 6.0 software,
1 þ RK e
then the content of various secondary structures of An-PEP was
Eb Pi calculated.
PF ¼  ð3Þ
Ei Pb
2.10.2. Fluorescence spectrometry anslysis
where Et, Eb and Ei are the enzyme activity (U/mL) of the top phase, bot- An-PEP concentrates were heated in various temperatures (50, 60,
tom phase and the crude extract, respectively. Pb and Pi are total protein 70, 80 and 90 °C) of water bath for 30 min and then measured using a
concentration (mg/mL) in the bottom and the crude extract, respec- fluorescence spectrophotometer for the fluorescence spectrum analysis.
tively. R is defined as the phase volume ratio. The emission spectrum scanning range was recorded within
300–460 nm at 280 nm excitation wavelength. Both the excitation slit
Vb and the emission slit width were 10 nm.
R¼ ð4Þ
Vt
2.10.3. Optimum temperature and temperature stability
where Vb and Vt are volumes of bottom phase and top phase,
The optimum temperature was determined by measuring the
respectively.
An-PEP concentrates activity in a range of 20–90 °C. Then the residual
enzyme activity of An-PEP was determined to evaluate its thermal sta-
2.8. Experimental design
bility. Besides, the An-PEP concentrates was incubated in phosphate
buffer (pH 5.0) at different temperature (20–90 °C) for 30 min to deter-
Through RSM experiment, the extraction conditions were opti-
mine the residual enzyme activity.
mized, and the interaction between parallel factors was investigated.
The effects of X1 ((NH4)2SO4 mass fraction), X2 (ethanol mass fraction)
2.10.4. Optimum pH and pH stability
and X3 (pH) were stuided. And the experiment took PF and Y of the bot-
The influence of pH on An-PEP concentrates activity was determined
tom phase as the responses. The experimental design was shown in
in various buffers at an optimal temperature (40 °C), including citrate
Table 1. Y and PF data were fitted with the interaction terms via a
buffer (0.05 mol/L) at pH 2.0–7.5, Tris-HCl buffer (0.05 mol/L) at
second-order polynomial equation model, as shown below:
pH 8.0–8.5 and Glycine-NaOH buffer (0.05 mol/L) at pH 9.0–10.0. Fi-
nally, the pH stability was estimated after incubating in buffers at
pH 2.0–10.0 for 30 min.
Table 1
Factors and levels in the response surface design used for ATPS optimization of separated
An-PEP. 2.10.5. Effect of metal ions on an-PEP activity
Different metal ions(Ca2+, K+, Fe2+, Mg2+, Zn2+, Mn2+, Cu2+, Fe3+)
Variables Coded variable levels
were added to An-PEP solutions made by An-PEP lyophilized powder to
−1 0 +1
5 mmol/L of each ion concentration. After 30 min, the activity of An-PEP
X1 (NH4)2SO4 (w/w)% 14 14.5 15 was measured. An-PEP solution without any metal ions was used as the
X2 Ethanol (w/w)% 26 27 28 100% relative active control. The 5 mmol/L colored metal ions (Cu2+,
X3 pH 5.5 6.0 6.5
Fe2+ and Fe3+) were used as corresponding blank controls.

386
B. Jiang, M. Wang, X. Wang et al.
2.10.6. Enzyme kinetics
The maximum reaction rate (Vmax) and the Michaelis–Menten con-
stant (Km) of An-PEP concentrats were determined using different con-
centrations (0.1–1 mM) of Ala-Ala-Pro-pNA as a substrate with the
standard method in Section 2.6 [30]. The kinetic parameters were calcu-
lated using Lineweaver – Burk plot.
2.11. Preparation of EWP hydrolysates
2.11.1. Hydrolysis of EWP with An-PEP
EWP was hydrolyzed as described by Norris [31]. EWP was dissolved
with ultrapure water to obtain 5% (w/v) EWP solution, which was
stirred at 40 °C for 30 min. And, the pH of EWP solution was adjusted
to 5.0 with HCl. Then, An-PEP lyophilized powder (PEP: EWP = 2.5%
(w/v)) was added to EWP solution to be incubated at 40 °C for different
hydrolysis times (1, 2, 3, 4, 8 and 24 h). After hydrolysis, the mixtures
were heated for 20 min at 80 °C to inactivate An-PEP. Finally, the mix-
tures were dialyzed against distilled water with 100 Da dialysis bag
for 24 h, then the dialyzates were concentrated and lyophilized to ob-
tain EWP hydrolysates powder.
2.11.2. Determination of degree of hydrolysis (DH) by TNBS method
According to the method described by Guan [32], the hydrolysis
conditions were slightly modified to define the conditions for enzy-
matic hydrolysis of EWP. Leucine was dissolved in 1% SDS solution
to prepare a 240 mg/mL standard solution. The standard solution
was diluted into 0, 60.0, 120.0, 180.0 and 240.0 mg/L leucine solu-
tions. 2 mL phosphate buffer, 2.0 mL of TNBS solution (0.1% (w/v)),
and 250 μL of leucine solution were mixed and incubated for
60 min in a 50 °C water bath. 4 mL of HCl (0.1 mol/L) was added to
the mixtures to stop the reaction. After 30 min, the absorbance at
340 nm of mixtures was determined. Then, the standard curve was
made by the above steps.
50 μL of EWP hydrolysates was diluted with 950 μL SDS (1% (w/v))
solution, which was heated for 5 min in 85 °C water bath to inactivate
An-PEP. 250 μL of the mixture was taken to determine its amino acid
content to obtain the DH of EWP. The DH was calculated according to
formula (6):
AN1 −AN2
DH% ¼  100 ð6Þ
Npb
where AN1 and AN2 are amino nitrogen content (mg/g protein) of EWP
before and after hydrolysis, respectively. Npb is nitrogen content (mg/g
protein) of peptide bonds in EWP.
2.11.3. Electrophoresis assay of EWP hydrolysates
The electrophoresis was made by the method of Wang et al. with
slightly modified [33]. EWP hydrolysates with different hydrolysis
times (1, 2, 3, 4, 8 and 24 h) were prepared into 10 mg/mL solutions
with ultrapure water. 12% bis-acrylamide homogeneous gel was used
in SDS-PAGE. Operation steps were the same as section
“Electrophoresis”.
2.12. Activity assay of EWP hydrolysates
2.12.1. OH• free radical scavenging activity
1 mL of hydrolysates solution (5 and 10 mg/L), 0.5 mL of salicylic
acid ethanol solution (9.0 mmol/L), 1.0 mL of FeSO 4 solution
(9.0 mmol/L) and 5 mL of ultrapure water were mixed. Thereafter,
1 mL of H2 O2 solution (8.8 mmol/L) was added to the mixtures.
Then, the reaction mixtures were incubated for 15 min at 37 °C.
The absorbance at 510 nm of the obtained solution was determined.
Ultrapure water replaced the hydrolysates solution as a control
group, and ultrapure water replaced the H2 O2 solution as a blank
387
International Journal of Biological Macromolecules 169 (2021) 384–395
group. Hydroxyl free radical scavenging activity was calculated as
formula (7):
AS −A0
OH: free radical scavenging ability ð%Þ ¼ 1−  100% ð7Þ
AC
where As, A0 and Ac are the absorbance of sample, the blank group and
the control group, respectively.
2.12.2. ABTS free radical scavenging ability
The ABTS free radical scavenging ability was measured by Jiang [34]
after slightly modified. 0.2 mL of 7 mmol/L ABTS and 0.2 mL of
2.6 mmol/L potassium persulfate solution were mixed and placed in
the darkness for 16 h to make an ABTS free radical stock solution.
Then, the ABTS free radical stock solution was diluted with phosphate
buffer solution (pH 7.4) to make the absorbance at 734 nm of 0.7 ±
0.02. Next, 0.6 mL of EWP hydrolysates solutions at different concentra-
tions (5 and 10 mg/L) were mixed with 2.4 mL of ABTS free radical dilu-
tion. And, the mixtures were incubated in the darkness for 6 min. After
using 95% ethanol as a control group, the absorbance at 734 nm of the
mixtures was determined. ABTS free radical scavenging ability was eval-
uated on the basis of formula (8):
Ac −As
ATBS free radical scavenging ability ð%Þ ¼  100% ð8Þ
Ac
where As and Ac are the absorbance of samples and the control group,
respectively.
2.12.3. Determination of ACE inhibitory activity
The ACE inhibitory activity was measured by Guan [32] after slightly
modified. 200 μL of 1 mg/mL EWP hydrolysate solution and 80 μL ACE
solution (25 mU/mL) were mixed and pre-incubated for 5 min at
37 °C. Then, 150 μL buffered substrate (5 mmol/L N-hippuryl-His-Leu
tetrahydrate in sodium borate buffer (50 mmol/L, pH 8.3) containing
0.3 mol/L NaCl) was added into the mixture, then, the obtained mixture
was incubated for 30 min at 37 °C and stopped with 1 mol/L HCl
(250 μL). 2 mL of ethyl acetate was mixed with the reaction solution
and then centrifuged at 2000 ×g for 10 min to obtain supernatant con-
taining hippuric acid. 1 mL of supernatant was placed in a vacuum con-
centrator until the solvent was completely evaporated. Finally, the
residue was dissolved with 3 mL ultrapure water and the absorbance
at 228 nm was measured with an ultraviolet spectrophotometer. Ultra-
pure water replaced EWP hydrolysates solution as a control group. A
sample with HCl first added to inhibit ACE activity was used as a blank
group. The ACE inhibitory activity (%) was calculated according to
formula (9):
AC −AS
ACE inhibitory activity ð%Þ ¼  100% ð9Þ
AC −A0
where AC AS and A0 are the absorbance of control group, samples and
blank group, respectively.
2.12.4. Determination of anti-gout activity
The anti-gout activity was determined by Johnson [35] after
slightly modified. Anti-gout activity was indicated by the inhibitory
activity of the sample on xanthine oxidase (XO). 30 μL of 1 mg/mL
EWP hydrolysates solution at different hydrolysis times (1, 2, 3, 4,
8 and 24 h), 21 μL of phosphate buffer (pH = 7.4) and 180 μL of
XO (0.5 U/mL) were added to a 96-well microtiter plate, and the
plate was placed for 15 min at 25 °C. Then, 36 μL of xanthine solu-
tion (1 mmol/L) was added. After reacting for 30 min at 25 °C,
150 μL of HCl (1 mol/L) was added to the mixture to stop the reac-
tion. The absorbance was measured at 290 nm by a microplate
reader. A sample of HCl added before XO was used as a blank
B. Jiang, M. Wang, X. Wang et al.
group. The calculation formula of XO inhibitory activity (%) was as
follows:
A−B
XO Inhibitory Activity ð%Þ ¼  100%
B Ke increased first and then decreased. As the (NH4)2SO4 mass fraction
where A and B are the absorbance of the blank group and samples,
respectively.
2.13. Analysis of statistical
All experiments were carried out in triplicate, and the data were
expressed as the mean ± standard deviation. Besides, all data were an-
alyzed by analysis of variance (ANOVA) to assess the obvious difference
between the means (p < 0.05).
3. Results and discussion
3.1. Analysis of single-factor variable
The results of Table S1 showed that the system composed of ammo-
nium sulfate and ethanol had the best extraction effect on An-PEP, so
ethanol and ammonium sulfate were used as the composition of ATPS
for further research. Effects of ethanol and (NH4)2SO4 mass fraction
and pH on the distribution of An-PEP were investigated. Based on Y, Ke
and PF, optimal conditions for separating and purifying An-PEP by
ATPS were studied.
The alcohol/salt ATPS was used to separate biomolecular substances
based on the “salting out” mechanism. Inorganic salt was a significant
Fig. 1. Comparison of single factor results. (a) The influence of the concentration of (NH4)2SO4 on the separation; (b) The influence of the concentration of ethanol on the separation; (c)
The influence of pH on the separation.
International Journal of Biological Macromolecules 169 (2021) 384–395
factor that affected the distribution behavior of protein. Between the
two phases in ATPS, the electrostatic potential difference and hydro-
philic interaction would be changed due to inorganic salts [36,37]. As
shown in Fig. 1a, as the (NH4)2SO4 mass fraction increased, PF, Y, and
ð10Þ
increased, the “salting out” effect the partitioning of protein,and the hy-
drophobicity and electrostatic repulsion also caused the protein solubil-
ity in the salt phase to decrease. Similar effects of (NH4)2SO4 mass
fraction were reported for the purification of bromelain purification
from pineapple processing waste [38]. When the (NH4)2SO4 mass frac-
tion was less than 14.5%, impurity protein gradually migrated to the top
phase, thereby Ke, PF and Y increased. When the mass fraction of (NH4)
2SO4 was above 14.5%, both An-PEP and impurity protein migrated to
the top phase and high ion concentration caused An-PEP to precipitate
out, which led to the decrease of Ke, PF and Y values. Thus, 14.5%
(NH4)2SO4 was more suitable for separation and purification of An-PEP.
Fig. 1b shows that, in the process of ethanol concentration optimiza-
tion, the partition coefficient of An-PEP in ATPS showed the same trend
as the (NH4)2SO4. As the ethanol mass fraction increases, PF and Ke in-
creased and then decreased. When the ethanol mass fraction was less
than 26%, the ATPS could not be formed. Until the mass fraction of eth-
anol reached 27%, PF, Ke and Y increased and the increase of PF was more
obvious than that of Y. It indicated that An-PEP was easier to be distrib-
uted in the bottom phase than impure proteins, which might be due to
the differences in molecular weight, shape, volume and surface area of
An-PEP and impure protein [39]. After the mass fraction of ethanol
exceeded 27%, PF, Y and Ke all decreased. As the ethanol mass fraction in-
creased excessively, the dehydration of ethanol caused some water
molecules to be transferred to the top phase. And the “salting out” effect
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B. Jiang, M. Wang, X. Wang et al. International Journal of Biological Macromolecules 169 (2021) 384–395

appeared in the bottom phase, which reduced the An-PEP content of the Table 3
bottom phase, thus resulting in the PF, Y and Ke of An–PEP were re- The variance analysis of a quadratic polynomial prediction model fitted to PF.

duced. With the increase of ethanol concentration, the distribution be- Source Sum of squares df Mean square F P1-value
havior in the ethanol/ammonium sulfate ATPS of An-PEP was similar Model 31.15 9 3.46 59.17 <0.0001
to that of the impurity proteins in the study of separation of ε-poly-L- Residual 0.41 7 0.058
lysine [40]. In summary, when the ethanol mass fraction was 27%, the Lack of fit 0.083 3 0.028 0.34 0.7985
maximum PF, Ke and Y of An-PEP in the bottom phase were obtained. Pure error 0.33 4 0.082
Cor total 31.56 16
pH was critical for ATPS to extract enzymes and proteins. Fig. 1c
CV(%) 1.11
shows the changes of PF, Ke and Y in the pH range of 5.0–7.0. At R21 0.9870
pH 5.0–6.0, PF, Ke and Y increased with the increase of pH. At
pH 6.0–7.0, PF decreased, while Ke and Y decreased first and then stabi-
lized after pH 6.5. As protein was amphoteric electrolyte whose charge
properties changed with pH, the pH affected the distribution of An- The absolute value of each coefficient in fitting equations could re-
PEP and impure protein in ATPS [41]. When the pH was overthe isoelec- flect the influence degree of each factor on response values. Quadratic
tric point of protein, protein was negatively charged and ion-repulsed coefficients of two fitting equations were negative values, indicating
with the sulfate ions in the bottom phase, prompting protein to migrate that equations had maximum points and could be optimized for analy-
to the top phase. When the pH was lower than the isoelectric point of sis. Linear coefficients of two equations indicated the order of factors af-
protein, protein was positively charged and more likely to stay in the fecting the PF was pH (X3) > ethanol mass fraction (X2) > ammonium
bottom phase. A similar observation was reported by Nan Shi [41], sulfate mass fraction (X1) and the order of factors affecting the Y were
working on recovery and partitioning of transglutaminase. In present pH (X3) > ammonium sulfate mass fraction (X1) > ethanol mass frac-
case, the An-PEP could be separated from other proteins, because the tion (X2).
impurity protein was mainly acidic amylase, and the isoelectric point
of acidic amylase (about 4.8–6) was different from An-PEP (4.43). 3.2.2. Analysis of variance
Therefore, when the pH was 6.0, PF, Ke and Y values reached maximum. The significance test of regression model for the response was eval-
According to the above, the best single-factor conditions could be got uated and the results of analysis of variance (ANOVA) tests were pre-
as follows: ATPS was made up of (NH4)2SO4 14.5% (w/w) and enthanol sented in Tables 3 and 4. P1-value and P2-value of model were less
27% (w/w) with pH 6.0. than 0.0001, which showed that the model could well describe the rela-
tionship between each single factor and response value. Fig. 2 shows
3.2. Analysis of the RSM that the correlation between predicted values and true values of PF
and Y prediction models was good. The correlation coefficients, R21 and
3.2.1. Statistical analysis and model fitting R22, were 0.9870 and 0.9741 of prediction models, respectively. It indi-
RSM was made to further study effects of (NH4)2SO4 mass fraction cated only 1.30% and 2.59% of PF and Y models could not be predicted.
(X1), ethanol mass fraction (X2) and pH (X3) on PF and Y. Total 17 exper- And coefficients of variation (CV) in this test were 1.11% and 2.38%, re-
iments (runs) were designed by Box–Behnken design (BBD) (Table 2) spectively. Results showed that the experimental data had high accu-
to obtaine the second-order prediction models with response values racy and reliability. Therefore, the model could be used for numerical
(PF and Y). And the obtained fitting equations were: fitting analysis.

PF ¼ 15:38–0:039X 1 þ 0:045X 2 –0:091X 3 –0:46X 1 X 2 þ 0:28X 1 X 3

3.2.3. Interactive analysis
þ 0:13X 2 X 3 –1:06X 1 2 In the three-dimensional (3D) response surface diagram, the rela-
tionship could be intuitively seen between the response of each variable
–1:92X 2 2 –1:26X 3 2 ð11Þ and the experimental levels, which offered an approach for directly ob-
serving the interaction in two experimental variables.The factors affect-
Y ¼ 89:47 þ 2:49X 1 þ 2:03X 2 þ 3:69X 3 þ 2:86X 1 X 2 þ 2:12X 1 X 3 ing PF and Y were displayed in 3D response surface plot in Fig. 3.
þ 2:15X 2 X 3 –5:78X 1 2 – The 3D response surface plots of the interaction between (NH4)2SO4
and ethanol on PF and Y values were shown in Fig. 3a and b, respec-
6:09X 2 2 –8:72X 3 3 ð12Þ tively. As the (NH4)2SO4 mass fraction and ethanol increased, PF and Y
increased and then decreased. It could be deduced that the hydrophobic
Table 2 interaction and weak ionic strength were not conducive to protein mi-
BBD and the results (means of triplicate tests) for the protein purification and recovery gration to the top phase, and both An-PEP and impurity protein
factor of An-PEP. remained in the bottom phase. High concentrations of (NH4)2SO4 and
Number X1 (NH4)2SO4 X2 ethanol X3 Purification Recovery ethanol increased ionic strength and hydrophobicity, which promoted
(w/w) % (w/w) % pH Factor (%) protein distribution to the top phase or precipitation at the interface
2 −1 0 1 12.74 74.41 of two phases. Therefore, low concentrations of (NH4)2SO4 and ethanol
3 −1 1 0 12.96 75.36 were not suitable for the separation of PEP and other protein.
4 1 0 −1 12.84 71.29
5 1 1 0 12.07 85.63
6 1 -1 0 12.76 74.11
7 -1 -1 0 12.06 74.46 table 4
8 0 -1 1 11.6 64.46 The variance analysis of a quadratic polynomial prediction model fitted to Y.
9 0 0 0 15 90.58
Source Sum of squares df Mean square F P2-value
10 1 0 1 13.11 84.04
11 0 0 0 15.53 90.12 Model 946.29 9 105.14 29.25 <0.0001
12 -1 0 -1 13.58 70.12 Residual 25.16 7 3.59
13 0 -1 -1 12.4 72.52 Lack of fit 8.92 3 2.97 0.45 0.5845
14 0 0 0 15.18 90.55 Pure error 16.24 4 4.06
15 0 0 0 15.51 85.88 Cor total 971.45 16
16 0 0 0 15.96 90.2 CV(%) 2.38
17 0 1 -1 12.09 70.56 R22 0.9741

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Fig. 2. Relationships between predicted and actual values in the models of PF (a) and Y (b).

Fig. 3c and d shows the interaction between the (NH4)2SO4 mass increased with increasing pH and the (NH4)2SO4 mass fraction,
fraction and pH on PF and Y, the trends of which were similar to resulting in more hydrophobic groups of impure protein exposed on
Fig. 3a and b. Higher or lower (NH4)2SO4 mass fraction and pH were the molecular surface. Thus, impure protein was distributed to the hy-
harmful to the separation of PEP in the bottom phase. The ionic strength drophobic ethanol phase (top phase), while An-PEP remained in the

Fig. 3. The plots of response surface for PF (a, c, e) and Y (b, d, f) of An-PEP.

390
B. Jiang, M. Wang, X. Wang et al.
Fig. 4. The effect of temperature on the activity and stability of purified An-PEP.
salt phase (bottom phase). Moreover, excessive ionic strength increased
the salting-out effect and An-PEP also moved to the ethanol phase,
which led to the distribution of impure protein and An-PEP to the top
phase, thereby PF and Y reduced.
The interaction between the ethanol mass fraction and pH on PF and
Y was indicated in Fig. 3e and f. As the ethanol mass fraction and pH in-
creased, Y and PF increased and then decreased. The effect of pH was re-
lated to the isoelectric point of An-PEP and impurity protein. When pH
was higher than the isoelectric point, protein were negatively charged.
Therefore, the protein repeled the negatively charged sulfate anion,
making the protein migrate to the upper phase. In contrast, when pH
was below the isoelectric point, the protein migrated to the bottom
phase. Due to the ethanol mass fraction could affect the hydrophobicity
of ATPS, the separation and purification of An-PEP was influenced by the
ethanol mass fraction. Moreover, after the ethanol mass fraction in-
creased to a certain value, An-PEP might be precipitated, resulting in a
decrease in An-PEP recovery.
3.2.4. Validation of the best extraction conditions
After optimizing conditions of ethanol/(NH4)2SO4 ATPS separation
of An-PEP by RSM, the predicted optimal separation conditions were
that: ethanol mass fraction was 27.08%, (NH4)2SO4 mass fraction was
14.56% and pH was 6.06. Under this condition, the predicted PF value
was 15.33 and the predicted Y value was 90.19%. The predicted condi-
tions had been rationalized and modified to facilitate experimental op-
erations: the (NH4)2SO4 mass fraction was 14.5%, the ethanol mass
fraction was 27% and pH was 6.0. After experimental verification, puri-
fication factor of An-PEP was 15.35 ± 0.30 and recovery of the An-PEP
was 90.29 ± 0.23%. There was no significant difference from the pre-
dicted PF and Y, indicating that optimization results of response surface
experiment were better. This extraction effect was higher than that of N.
Shi [41], Agrawal [43], Siqueira [44] and Silva [45] using ATPS for prote-
ase, and also more simple than Kang [30] used 10 kDa ultrafiltration
Table 5
The content of the secondary structure of An-PEP incubated at different temperatures.
Secondary structure β-sheet (%)
Untreated 39.44 ± 0.35c
50 °C 39.25 ± 0.29b
60 °C 39.05 ± 0.27a
70 °C 39.46 ± 0.32d
80 °C 40.22 ± 0.37f
90 °C 39.85 ± 0.28e
Note: The same letter in the same column indicates that the difference is not significant (p > 0.05) and the difference of the marked letters indicates that the difference is significant
(p < 0.05).
International Journal of Biological Macromolecules 169 (2021) 384–395
membrane to purify aspergillus oryzae PEP and Yu [5] used Ni-
Sepharose and ÄKTA purifier system with a Superose column to purified
a prolyl endopeptidase from Stenotrophomonas maltophilia.3.3. Electro-
phoretic analysis.
Results of SDS-PAGE were shown in Fig. S1. There were three bands,
a, b and c, in the crude extract (lane 2). The top phase of ATPS (lane
4) showed b and c bands and the bottom phase of ATPS (lane 3) had a
and b bands, which indicated that the used ATPS had good selectivity.
Band a in the bottom phase of ATPS was An-PEP with a molecular
weight of 66–116 kDa. Because An-PEP was a highly glycosylated pro-
tein, its electrophoretic band presented a diffuse state and the molecular
weight was not a constant value but a range. In addition, bands b and c
might be amylases. To be more specific, band b was an acid-stable am-
ylase and band c was an acid-labile amylase. At low pH, acid labile am-
ylase might degrade. Results of SDS-PAGE showed that the molecular
weight of the purified An-PEP was in the range of 66–116 kDa, and eth-
anol/(NH4)2SO4 ATPS achieved the effect of separation and purification
of An-PEP.
3.3. Properties of An-PEP
3.3.1. Optimum temperature and temperature stability
The results were shown in Fig. 4. From the activity results, the en-
zyme activity of An-PEP increased at 20–40 °C and reached its maxi-
mum at 40 °C with the increase of temperature. It decreased slightly
at 40–70 °C, and then decreased sharply at 70–90 °C. An-PEP could
maintain more than 90% of the enzyme activity at 35–70 °C. From the
thermal stability results, the enzyme still retained more than 80% of its
initial activity after incubating at 25 °C–50 °C for 30 min. While only
about 30% and below 10% of the enzyme activity was retained at 55 °C
and 65 °C, respectively, and An-PEP was inactivated after incubating at
85 °C for 30 min. Therefore, combining the two results, 40 °C was the
optimal temperature for An-PEP. A similar observation was reported
by Kang [30]. And the optimal temperature of An-PEP was also similar
to the PEP from A. niger (42 °C) [7] and Pseudomonas sp. KU-22
(45 °C) [1].
The effect of temperature on the secondary structure of An-PEP was
studied by Fourier infrared spectroscopy (FTIR). The original spectrum
of the amide Ι band (1600 cm−1 − 1700 cm−1) of An-PEP treated at dif-
ferent temperature (50, 60, 70, 80, and 90 °C) was used to calculate the
second derivative, and the overlapped peaks of amide I was fitted by the
Gauss peak to show the content of secondary structure. The correspond-
ing relation between each secondary structure and sub-peak were
1650–1660 cm−1 of α-helix; 1610–1640 cm−1 of β-sheet;
1660–1700 cm−1 of β-turn and 1640–1650 cm−1 of unordered. Results
were shown in Table 5. The α-helix content decreased slightly in general
with the temperature increased, while the random coil content in-
creased. It indicated that the high temperature damaged An-PEP struc-
ture, resulting in a decrease in its activity.
In general, the conformational changes of protein could also be stud-
ied by fluorescence spectroscopy [54]. The fluorescence changes of An-
PEP handled at different temperatures were shown in Fig. 5. After heat
treatment at 50 °C, there was no obvious change in the fluorescence
Random coil (%) α-Helix (%) β-turn (%)
17.06 ± 0.18a 15.66 ± 0.14e 27.84 ± 0.23c
17.08 ± 0.25b 15.60 ± 0.21d 28.07 ± 0.27e
17.31 ± 0.12c 15.44 ± 0.32b 28.20 ± 0.23f
17.49 ± 0.22b 15.11 ± 0.21c 27.95 ± 0.31d
17.68 ± 0.17d 15.09 ± 0.17d 26.61 ± 0.12a
17.73 ± 0.15e 14.64 ± 0.11a 27.78 ± 0.29b
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B. Jiang, M. Wang, X. Wang et al.
Fig. 5. Fluorescence spectroscopy of An-PEP treated at different temperatures.
intensity and maximum emission wavelength of An-PEP. However, the
maximum emission wavelength of An-PEP was red-shifted, when An-
PEP was treated at 60 °C to 90 °C. The red shift might be due to the
greater exposure of tryptophan residues to the solvent, which changed
the hydrophobic environment of the tryptophan residues. In addition,
the fluorescence of An-PEP treated at 80 °C and 90 °C was quenched.
It meant that excessive temperature would destroy the tertiary struc-
ture of An-PEP. This also further indicated that An-PEP had good ther-
mal stability under the condition of less than 50 °C. And when the
temperature was higher than 50 °C, the structure of An-PEP was
destroyed, causing its thermal stability to decrease.
3.3.2. Optimum pH and pH stability
The optimum pH for purified An-PEP was 4.5–5.0 (Fig. 6), which in-
dicated that An-PEP was a type of acid protease. An-PEP was placed in
buffers of different pH for 30 min, and then the pH stability was investi-
gated by measuring the enzyme activity of An-PEP. An-PEP remained
more than 80% activity after incubation between pH 3.5 and 6.0 for
30 min, indicating that An-PEP had good stability in the range of
pH 3.5–6.0.
3.3.3. Effect of metal ions on An-PEP activity
5 mmol/L different metal ion solutions (Ca2+, K+, Fe2+, Mg2+, Zn2+,
Mn2+, Cu2+ and Fe3+) were used to investigate the effect of metal ions
Fig. 6. The effect of pH on the purified An-PEP activity and stability.
392
International Journal of Biological Macromolecules 169 (2021) 384–395
on An-PEP activity. Results were shown in Fig. S2. Ca2+ exhibited an ac-
tivation effect on An-PEP activity and the similar result was obtained for
the PEP from Aspergillus oryzae [3,30]. K+ did not show any effects on
An-PEP activity, while other metal ions such as Fe2+, Mg2+, Zn2+,
Mn2+, Cu2+ and Fe3+ exhibited inhibition effects on An-PEP activity.
After treated by Fe2+, Mg2+, Zn2+, Mn2+ and Cu2+, An-PEP activity
could retained more than 80%, whereas Fe3+ showed stronger An-PEP
activity inhibition than other metal ions.
An-PEP treated by Ca2+ and Fe3+ ions and untreated An-PEP were
analyzed by infrared fitting. The secondary structure analysis was indi-
cated in Table 6. The results showed that Ca2+ increased the β-sheet
content and decreased the β-turn content of An-PEP, while Fe3+ re-
duced the β-sheet content and increased the β-turn content of An-
PEP, and also reduced the random coil and α-helix a little bit. It indicated
that the activation and inhibition of An-PEP activity by metal ions might
be related to β-sheet and β-turn structure of An-PEP.
3.3.4. Enzyme kinetics
The activity of An-PEP was determined by the substrate which con-
tains a chromophore p-nitroaniline. The chromophore combined with
the carboxyl group of Pro through its amino group to form an amide
bond. And this peptide bond on the carboxyl side of Pro was hydrolyzed
by An-PEP to generate benzyloxycarbonyl -dipeptide and p-nitroaniline
[46]. Kinetic data of the An-PEP concentrats were evaluated at 40 °C and
pH 5.0. Using Ala-Ala-Pro-pNA as substrates, the catalytic reaction rate
(V) of An-PEP was determined at different concentrations ([S]). Using
double reciprocal plotting, 1/[S] and 1/V were plotted as the horizontal
and vertical coordinates, respectively. Results were shown in Fig. S3.
The obtained fitting curve equation was 1/V = 0.1373/[S] + 0.275
(R2 = 0.9947). It was concluded that Km and Vmax of An-PEP were
0.5 mmol/L and 3.64 μmol/(L•min), respectively. It indicated that the
An-PEP had a high affinity with Ala-Ala-Pro-pNA. A similar result was
obtained for the proly endopeptidase from A. oryzae S1 [3].
3.4. Preparation of EWP hydrolysates
3.4.1. Determination of DH
The number of amino groups released by breaking the peptide bonds
was quantified as the percentage of DH. The standard curve equation
measured by the experiment was y = 0.0017× + 0.0064 (R2 =
0.999), and the DH of EWP at different hydrolysis times were showed
in Table 7. DH represented the percentage of hydrolyzed peptide
bonds in the protein to total peptide bonds, hence the length of the pep-
tide chain was related to DH. A low DH indicated a long peptide chain
and a high DH indicated a short peptide chain. With the extension of hy-
drolysis time, DH of EWP gradually increased, indicating that the pep-
tide chain of EWP hydrolysate would be shorter.
3.4.2. SDS-PAGE
As shown in Fig. 7, bands in the hydrolysates obtained at 3 h hydro-
lysis time in lane 5 were significantly shallower than bands in other
lanes. The degree of hydrolysis results showed that DH of hydrolysates
made by hydrolyzing in 3 h was not the highest. For other hydrolysates,
as the hydrolysis time prolonged, bands of EWP between 66.2 and
116.0 kDa gradually became shallower and that between
45.0–66.2 kDa and 35.0–45.0 kDa gradually deepened. It indicated
that proteins of 66.2–116.0 kDa were hydrolyzed by An-PEP into pep-
tides with molecular weight of 35.0–66.2 kDa. Bands between 66.2
and 116.0 kDa were the shallowest and bands between 45.0 and
66.2 kDa were the deepest in the hydrolysates for 24 h hydrolyzing. In
addition, bands of hydrolysates less than 14.4 kDa in the hydrolysates
became lighter, which showed that protein had been hydrolyzed into
smaller molecular weight peptides. It further showed that DH of hydro-
lysates for 24 h became higher than that of hydrolyzed for less than 24 h,
which was the similar as the measured result of DH. Ovotransferrin and
lysozyme in egg white protein were allergens, so An-PEP hydrolyzed
B. Jiang, M. Wang, X. Wang et al. International Journal of Biological Macromolecules 169 (2021) 384–395

Table 6
The effect of metal ions on An-PEP activity.

Secondary structure β-sheet (%) Random coil (%) α-Helix (%) β-Turn (%)

Untreated 39.44 ± 0.35b 17.06 ± 0.18b 15.66 ± 0.14b 27.84 ± 0.23b

Ca2+ 40.38 ± 0.27c 17.95 ± 0.21c 16.22 ± 0.18c 25.44 ± 0.19a
Fe3+ 38.62 ± 0.23a 16.50 ± 0.14a 15.50 ± 0.21a 29.39 ± 0.26c

Note: The same letter in the same column indicates that the difference is not significant (p > 0.05) and the difference of the marked letters indicates that the difference is significant
(p < 0.05).

EWP could reduce its sensitization. And 24 h hydrolysates might have
the lower sensitization.
3.5. Activity assay of EWP hydrolysates
3.5.1. Determination of antioxidant activity
This part of experiments was to evaluate antioxidant activities of
EWP hydrolysates at different times through OH· and ABTS free radical
scavenging ability.
Hydroxyl and their derivative radicals were high-efficiency oxidants,
which could interact with most biological macromolecules in living cells
to cause serious biological damage and lipid peroxidation [47]. In the
presence of metal ions such as copper or iron, hydroxyl radicals could
be formed from superoxide anions and hydrogen peroxide. When the
hydroxyl radical reacted with an aromatic compound, it could add to a
double bond to form the hydroxyl cyclohexadienyl group, as salicylic
acid reacted with OH· to form 2, 5-dihydroxy benzoic acid and 2, 3-
dihydroxy benzoic acid. As shown in Fig. 8a, there was no linear rela-
tionship between hydroxyl radical scavenging ability and hydrolysis
time. EWP hydrolysates obtained at 1 h hydrolysis time had the stron-
gest hydroxyl radical scavenging ability, followed by the 3 h EWP hydro-
lysates, and there was not much difference in hydroxyl radical
scavenging ability of EWP hydrolysates for 2, 4, 8 and 24 h hydrolyzing.
ABTS was oxidized by K2S2O8 into blue-green ABTS free radicals that
were stable in water. When it reacted with antioxidants, the blue-green
color would became lighter. Therefore, when a sample with strong anti-
oxidant activity was present, the reaction solution would show a lighter
color. ABTS free radical scavenging ability of EWP hydrolysates was
shown in Fig. 8b. The hydrolysis time was not directly proportional to
the ABTS free radical scavenging ability of EWP hydrolysates. EWP hy-
drolysates made by hydrolyzing for 1 and 24 h exhibited strong antiox-
idant activity, which might be related to the relative molecular mass,
amino acid sequence and amino acid side chain groups of EWP
hydrolysates.
3.5.2. Determination of ACE inhibitory activity
ACE inhibitory activity of EWP hydrolysates which was hydrolyzed
with An-PEP for 1–24 h was shown in Table 8. At 1–4 h, ACE inhibitory
activity gradually increased and reached its maximum at about 4 h, in-
dicating that as the hydrolysis time lengthened, more and more ACE in-
hibitory peptides were produced. At 4–8 h, the ACE inhibitory activity
decreased, this might be because ACE inhibitory peptides obtained in
about 4 h hydrolysis were enzymatically hydrolyzed into small peptides
without ACE inhibitory activity. The increase ACE inhibitory activity of
the hydrolysates at 24 h might owing to the production of other ACE in-
hibitory peptides after a longer hydrolysis time [48]. ACE inhibitory ac-
tivity of sodium caseinate hydrolysates, produced by Lactobacillus
helveticus at different incubation times, reached a similar conclusion,
which found that the EWP hydrolysatesfor 1 h and 2 h hydrolyzing
reached the maximum ACE inhibitory activity and the hydrolysates of
5 h greatly decreased [49]. And consistent with the results of Mullally
[50], the result suggested that when the hydrolysis exceeded a certain
level, the biological activity of peptides in cheese would decrease. It
was also found that there were no significant difference in ACE IC50
values of β-casein hydrolysates which were got in 4 and 24 h of hydro-
lysis [31]. In addition, combining the results of DH, it showed that the in-
crease in DH was not always linearly related to the increase in ACE
inhibitory activity. Some studies reported that ACE inhibitory activity
of hydrolysates was not entirely attributed to the DH and might be
due to the amino acid composition of peptides presented in hydroly-
sates, which depended on the used enzyme and protein [51].
3.5.3. Determination of anti-gout activity
Hyperuricemia was a gout-related disease and caused by excessive
secretion or insufficient excretion of uric acid. Gout was largely affected
by the intake of nucleic acid-rich foods in the diet [52]. XO (EC 1.2.3.2)
could catalyze xanthine to produce uric acid, so XO was a target for
the treatment of gout drugs. Therefore, researchers established an
in vitro screening platform for XO to study anti-gout activity by measur-
ing inhibitory activity and animal experiments [53]. Allopurinol and fla-
vonoids could effectively inhibit the activity of xanthine oxidase.
However, the anti-gout activity of peptides was still in its infancy. The

Table 7
The DH values of EWP treated with different hydrolysis
times.

Time(h) DH(%)

1 0.97 ± 0.17a
2 1.45 ± 0.21b
3 2.19 ± 0.27c
4 2.67 ± 0.24d
8 4.61 ± 0.18e
24 6.06 ± 0.30f

Note: The DH values are shown in the form of mean ±

standard deviation, and these values are the means mea-
sured in triplicate of the hydrolyzed samples. The same
letter followed by the same column means that the dif-
ference is not significant (p > 0.05), and the difference
between marked letters indicates that the difference is Fig. 7. SDS-PAGE electrophoresis analysis of the EWP hydrolysates obtained at different
significant (p < 0.05). hydrolysis times.(1: Marker; 2: EWP; 3: 1 h; 4: 2 h; 5: 3 h; 6: 4 h; 7: 8 h; 8: 24 h;)

393
B. Jiang, M. Wang, X. Wang et al.
Fig. 8. OH· (a) and ABTS (b) free radical scavenging ability of EWP hydrolysates obtained at different hydrolysis times.
Table 8
The ACE inhibitor activity of EWP hydrolysates obtained at different
hydrolysis times.
Time(h) ACE inhibitory activity (%)
1 26.19 ± 0.42b
2 28.57 ± 1.82c
3 30.95 ± 1.38d
4 45.24 ± 1.58f
8 25.00 ± 0.71a
24 32.14 ± 0.61e
Note: The ACE inhibitory activity values are shown in the form of
mean ± standard deviation, and these values are the means measured
in triplicate of the hydrolyzed samples. The same letter followed by the
same column means that the difference is not significant (p > 0.05),
and the difference between marked letters indicates that the difference
is significant (p < 0.05).
experiment examined the anti-gout activity of EWP hydrolysates
achieved by hydrolysis at various times. Table 9 shows that the XO in-
hibitory activity of EWP hydrolysates at different hydrolysis times was
significantly different. Inhibitory activities of 3 h and 4 h EWP hydroly-
sates on XO were 14.07 ± 0.33% and 15.14 ± 0.24%, and were stronger
than other hydrolysates. It indicated that 3 h and 4 h EWP hydrolysates
had relatively high anti-gout activity.
4. Conclusions
Although different methods have been used to isolate PEP from a
large variety of organisms, they were inferior to ATPS that was high re-
covery rate, low protein denaturation and cost-effective. ATPS
Table 9
The anti-gout activity of EWP hydrolysates obtained at different hy-
drolysis times.
Time(h) XO inhibitory activity (%)
1 11.82 ± 0.65d
2 7.64 ± 0.31c
3 3.59 ± 0.35a
4 14.07 ± 0.33e
8 15.14 ± 0.24f
24 3.94 ± 0.38b
Note: The same letter followed by the same column means that the
difference is not significant (p > 0.05), and the difference between
marked letters indicates that the difference is significant (p < 0.05).
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International Journal of Biological Macromolecules 169 (2021) 384–395
composed by ethanol and (NH4)2SO4 was successfully used to separate
An-PEP from Aspergillus niger fermentation broth. The optimal ATPS re-
action conditions were got by RSM and a BBD based on single-factor ex-
periment. The optimal conditions for ATPS were as follows: 14.5% (w/w)
(NH4)2SO4, 27% (w/w) ethanol and pH 6.0. Under these conditions, pu-
rification factor and recovery of An-PEP were 15.35 ± 0.30 and 90.29 ±
0.23%, respectively. It showed that ATPS could be used as a more easier,
efficient and valuable neomethod than other methods for the separa-
tion of An-PEP from fermentation broth in laboratory and industrial
processes. The molecular weight of An-PEP was 60–116 kDa and the op-
timum pH and temperature of An-PEP were 4.5–5 and 40 °C, respec-
tively. In addition, An-PEP could be applied to hydrolyze EWP, thus
reducing the sensitization of EWP and producing active peptides. The
results of electrophoresis analysis showed that the best DH of An-PEP
to EWP was at 24 h, and the results of the activity analysis of EWP hy-
drolysates showed that the obtained EWP hydrolysates had good scav-
enging ability of OH• and ABTS free radicals, ACE inhibitory activity and
the anti-gout activity. Current work suggests that a low-cost and high-
efficiency An-PEP separation technology was provided, and a theoretical
basis also was provided for studying the biological activity of egg white
protein peptides.
CRediT authorship contribution statement
Bin Jiang: Conceptualization, Methodology Supervision, Supervision,
Data curation
Meichan Wang: Conceptualization, Methodology, Software Writing-
Original draft preparation and Editing
Xiaojing Wang: Visualization, Investigation, Data curation
Shuang Wu: Supervision, Writing- Reviewing and Editing
Dongmei Li: Visualization, Investigation, Data curation
Chunhong Liu: Software, Validation, Data curation
Zhibiao Feng: Conceptualization, Methodology, Supervision and
Editing
Jie Li: Supervision, Writing- Reviewing and Editing
Declaration of competing interest
The authors have declared no conflicts of interest.
Acknowledgments
The authors would like to acknowledge the financial support from
the Natural Science Foundation of Heilongjiang Province (C2018019).
B. Jiang, M. Wang, X. Wang et al.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijbiomac.2020.12.120.
References
[1] H. Oyama, H. Aoki, M. Amano, E. Mizuki, T. Yoshimoto, D. Tsuru, S. Murao, Purifica-
tion and characterization of a prolyl endopeptidase from Pseudomonas sp. KU-22, J.
Ferment. Bioeng. 84 (6) (1997) 538–542.
[2] X. Li, Y. Cong, M. Ma, Z. You, B. Gao, J.Z.H. Zhang, L. Zhang, An energy optimization
strategy based on the perfect conformation of prolyl endopeptidase for improving
catalytic efficiency, J. Agric. Food Chem. 68 (2020) 5129–5137.
[3] C. Kang, X.Y. Yu, Y. Xu, Cloning and expression of a novel prolyl endopeptidase from
Aspergillus oryzae and its application in beer stabilization, J. Ind. Microbiol.
Biotechnol. 42 (2015) 263–272.
[4] W.Y. Li, Y. Li, Y.L. Chen, J.J. Hu, H.M. Mengist, G.M. Liu, T. Jin, M.J. Cao, Characteriza-
tion and crystal structure of prolyl endopeptidase from abalone (Haliotis discus
hannai), Food Chem. 333 (2020), 127452, .
[5] J. Yu, J. Wu, D. Xie, L. Du, Y.J. Tang, J. Xie, D. Wei, Characterization and rational design
for substrate specificity of a prolyl endopeptidase from Stenotrophomonas
maltophilia, Enzyme. Microb. Tech. 138 (2020), 109548, .
[6] R. Norris, A. Poyarkov, M.B. O’Keeffe, R.J. FitzGerald, Characterisation of the hydro-
lytic specificity of Aspergillus niger derived prolyl endoproteinase on bovine β-
casein and determination of ACE inhibitory activity, Food Chem. 156 (2014) 29–36.
[7] L. Edens, P. Dekker, R. van der Hoeven, F. Deen, A. de Roos, R. Floris, Extracellular
prolyl endoprotease from Aspergillus niger and its use in the Debittering of protein
hydrolysates, J. Agric. Food Chem. 53 (20) (2005) 7950–7957.
[8] I. Benucci, M.C. Caso, T. Bavaro, S. Masci, M. Keršienė, M. Esti, Prolyl endopeptidase
from Aspergillus niger immobilized on a food-grade carrier for the production of
gluten-reduced beer, Food Control 110 (2019), 106987, .
[9] B.V. Mohan Kumar, S. Swati, P. Prabhasank, Targeted degradation of gluten proteins
in wheat flour by prolyl endoprotease and its utilization in low immunogenic pasta
for gluten sensitivity population, J. Cereal Sci. 87 (2019) 59–67.
[10] L. Tsiatsiani, M. Akeroyd, M. Olsthoorn, A.J.R. Heck, Aspergillus niger prolyl
endoprotease for hydrogen–deuterium exchange mass spectrometry and protein
structural studies, Anal. Chem. 89 (15) (2017) 7966–7973.
[11] M. Wadhawan, S. Tiwari, S. Sharma, S. Rathaur, Identification and characterization of
a novel prolyl oligopeptidase in filarial parasite Setaria cervi, BBRC 495 (2018)
2235–2241.
[12] Y. Wang, F. Wang, S. Xu, R. Wang, C. Tian, Y. Ji, Q. Yang, Z.Q. Ping, Q. Xia, Transdermal
peptide conjugated to human connective tissue growth factor with enhanced cell
proliferation and hyaluronic acid synthesis activities produced by a silkworm silk
gland bioreactor, Appl. Microbiol. Biotechnol. 104 (23) (2020) 9979–9990.
[13] P.L. Show, C.P. Tan, M. Shamsul Anuar, A. Ariff, Y.A. Yusof, S.K. Chen, T.C. Ling, Extrac-
tive fermentation for improved production and recovery of lipase derived from
Burkholderia cepacia using a thermoseparating polymer in aqueous two-phase sys-
tems, Bioresour. Technol. 116 (2012) 226–233.
[14] W.N. Phong, P.L. Show, Y.H. Chow, T.C. Ling, Recovery of biotechnological products
using aqueous two phase systems, J. Biosci. Bioeng. 126 (3) (2018) 273–281.
[15] Y.K. Leong, P.L. Show, J.C.W. Lan, R. Krishnamoorthy, D.T. Chu, D. Nagarajan, H. Yen,
J.S. Chang, Application of thermo-separating aqueous two-phase system in extrac-
tive bioconversion of polyhydroxyalkanoates by Cupriavidus necator H16, Bioresour.
Technol. 287 (2019), 121474, .
[16] S.R. Chia, K.W. Chew, P.L. Show, A. Xia, S.H. Ho, J.W. Lim, Spirulina platensis based
biorefinery for the production of value-added products for food and pharmaceutical
applications, Bioresour. Technol. 289 (2019), 121727, .
[17] F. Chikari, J. Han, Y. Wang, P. Luo, X. He, E. Kwaw, P. Otu, Dual-frequency ultrasound-
assisted alcohol/salt aqueous two-phase extraction and purification of Astragalus
polysaccharides, J. Food Process Eng. 43 (4) (2020), e13366, .
[18] P.L. Show, C.W. Ooi, M.S. Anuar, A. Ariff, Y.A. Yusof, S.K. Chen, M.S.M. Annuar, T.C.
Ling, Recovery of lipase derived from Burkholderia cenocepacia ST8 using sustainable
aqueous two-phase flotation composed of recycling hydrophilic organic solvent and
inorganic salt, Sep. Purif. Technol. 110 (2013) 112–118.
[19] A.C.R. Caldeira, W.F.L. Franca, A. Converti, W.J.N. Lima, F.C. Sampaio, J.T. Faria, Liquid-
liquid equilibria in aqueous two-phase ethanol/salt systems at different tempera-
tures and their application to anthocyanins extraction, Food Sci. Technol. 39
(2019) 711–717.
[20] P. Tong, L. Gao, J. Gao, X. Li, Z. Wu, A. Yang, H. Chen, Iron-induced chelation alleviates
the potential allergenicity of ovotransferrin in a BALB/c mouse model, Nutr. Res. 47
(2017) 81–89.
[21] S. Lin, Y. Jin, M. Liu, Y. Yang, M. Zhang, Y. Guo, J. Gregory, Y. Yin, Research on the
preparation of antioxidant peptides derived from egg white with assisting of
high-intensity pulsed electric field, Food Chem. 139 (1–4) (2013) 300–306.
[22] J. Sun, H. Jing, Y. Mu, D.J. McClements, S. Dong, B. Xu, Fabrication of antioxidant
emulsifiers from natural ingredients: conjugation of egg white proteins with cate-
chin and chlorogenic acid, Food Hydrocoll. 108 (2020), 106019, .
[23] Y. Kobayashi, P. Rupa, J. Kovacs-Nolan, P.V. Turner, T. Matsui, Y. Mine, Oral Adminis-
tration of hen egg White Ovotransferrin Attenuates the development of colitis in-
duced by dextran sodium sulfate in mice, J. Agric. Food Chem. 63 (2015) 1532–1539.
[24] T. Rommanee, C.L. Deng, Antioxidant and antimicrobial activities of different enzy-
matic hydrolysates from desalted duck egg white, Asian-Australas J. Anim. Sci. 33
(9) (2020) 1487–1496.
395
International Journal of Biological Macromolecules 169 (2021) 384–395
[25] J. Yuan, Y. Zheng, Y. Wu, Double enzyme hydrolysis for producing antioxidant pep-
tide from egg white: optimization, evaluation, and potential allergenicity, Food
Biochem 44 (2) (2020), e13113, .
[26] R.B. Shashidhara, R. Iyyaswami, Aqueous two phase based selective extraction of
mannose/glucose specific lectin from Indian cultivar of Pisum sativum seed, J.
Chromatogr. B 1114 (2019) 13–23.
[27] T. Jiang, C. Kang, X.W. Yu, Y. Xu, High-level expression of prolyl endopeptidase in Pichia
pastoris using PLA2 as a fusion partner, J. Mol. Catal. B-Enzym. 125 (2016) 81–87.
[28] B. Jiang, J. Na, L. Wang, D. Li, C. Liu, Z. Feng, Reutilization of food waste: one-step
extration, purification and characterization of ovalbumin from salted egg white by
aqueous two-phase flotation, Foods 8 (8) (2019) 286.
[29] Y. Zhang, S. Liang, J. Zhang, Y. Chi, B. Tian, L. Li, B. Jiang, D. Li, Z. Feng, C. Liu, Prepa-
ration of whey protein isolate nanofibrils by microwave heating and its application
as carriers of lipophilic bioactive substances, LWT 125 (2020), 109213, .
[30] C. Kang, X.W. Yu, Y. Xu, Purifcation and characterization of a prolyl endopeptidase
isolated from Aspergillus oryzae, J. Ind. Microbiol. Biotechnol. 41 (2014) 49–55.
[31] R. Norris, A. Poyarkov, M.B. O’Keeffe, R.J. FitzGerald, Characterisation of the hydro-
lytic specificity of Aspergillus niger derived prolyl endoproteinase on bovine β-
casein and determination of ACE inhibitory activity, Food Chem. 156 (2014) 29–36.
[32] H. Guan, X. Diao, F. Jiang, J. Han, B. Kong, The enzymatic hydrolysis of soy protein
isolate by Corolase PP under high hydrostatic pressure and its effect on bioactivity
and characteristics of hydrolysates, Food Chem. 245 (2018) 89–96.
[33] Q. Wang, W. Liu, B. Tian, D. Li, C. Liu, B. Jiang, Z. Feng, Preparation and characteriza-
tion of coating based on protein nanofibers and polyphenol and application for
salted duck egg yolks, Foods. 9 (4) (2020) 449.
[34] B. Jiang, X. Wang, L. Wang, S. Wu, D. Li, C. Liu, Z. Feng, Fabrication and characteriza-
tion of microemulsion stabilized by integrated phosvitin and gallic acid, J. Agric.
Food Chem. 68 (19) (2020) 5437–5447.
[35] P. Johnson, C. Loganathan, A. Iruthayaraj, K. Poomani, P. Thayumanavan, S-allyl cys-
teine as potent anti-gout drug: insight into the xanthine oxidase inhibition and anti-
inflammatory activity, Biochimie 154 (2018) 1–9.
[36] J.A. Asenjo, B.A. Andrews, Aqueous two-phase systems for protein separation: a per-
spective, J. Chromatogr. A 1218 (49) (2011) 8826–8835.
[37] L. Dong, J.b. Wan, X. Cao, Separation of transglutaminase using aqueous two-phase sys-
tems composed of two pH-response polymers, J. Chromatogr. A 1555 (2018) 106–112.
[38] D.F. Coelho, E. Silveira, A. Pessoa Junior, E.B. Tambourgi, Bromelain purification
through unconventional aqueous two-phase system (PEG/ammonium sulphate),
Bioprocess Biosyst. Eng. 36 (2) (2012) 185–192.
[39] S. Dreyer, P. Salim, U. Kragl, Driving forces of protein partitioning in an ionic liquid-
based aqueous two-phase system, Biochem. Eng. J. 46 (2) (2009) 176–185.
[40] X.S. Chen, W.J. Diao, Y. Ma, Z.G. Mao, Extraction and purification ofε-poly-L-lysine
from fermentation broth using an ethanol/ammonium sulfate aqueous two-phase
system combined with ultrafiltration, RSC Adv. 10 (2020), 29587, .
[41] N. Shi, H. Xu, K. Guo, C. Kang, W. Zhang, Y. Zhang, L. Zhang, J. Tan, Extraction of mi-
crobial transglutaminase from Amycolatopsis sp. fermentation broth using aqueous
two-phase system, Korean J. Chem. Eng. 34 (8) (2017) 2248–2254.
[43] K. Agrawal, P. Verma, Production optimization of yellow laccase from Stropharia sp.
ITCC 8422 and enzyme-mediated depolymerization and hydrolysis of lignocellulosic
biomass for biorefinery application, Biomass Convers. Bior. (2020)https://doi.org/
10.1007/s13399-020-00869-w.
[44] J.G.W. Siqueira, T.M.S. Torres, B.M.V. Alves, A.L.F. Porto, T.S. Porto, Extraction of protease
from Aspergillus tamarii URM 4634 in aqueous two-phase system under continuous
and discontinuous process, Prep. Biochem. Biotechnol. 50 (6) (2020) 556–563.
[45] J. de C. Silva, P.R.L. de França, T.S. Porto, Optimized extraction of polygalacturonase
from Aspergillus aculeatus URM4953 by aqueous two-phase systems PEG/citrate, J.
Mol. Liq. 263 (2018) 81–88.
[46] N. Mika, H. Zorn, M. Rühl, Prolyl-specific peptidases for applications in food protein
hydrolysis, Appl. Microbiol. Biotechnol. 99 (19) (2015) 7837–7846.
[47] H.X. Yang, J.J. Deng, Y. Yuan, Two novel exopolysaccharides from Bacillus
amyloliquefaciens C−1: antioxidation and effect on oxidative stress, Curr. Microbial.
70 (2015) 298–306.
[48] R. Norris, M.B. O’Keeffe, A. Poyarkov, R.J. FitzGerald, Peptide identification and angio-
tensin converting enzyme (ACE) inhibitory activity in prolyl endoproteinase digests
of bovine αs-casein, Food Chem. 188 (2015) 210–217.
[49] M.C. Robert, A. Razaname, M. Mutter, M.A. Juillerat, Identification of angiotensin-I-
converting enzyme inhibitory peptides derived from sodium caseinate hydrolysates
produced by Lactobacillus helveticus NCC 2765, J. Agric. Food Chem. 52 (23) (2004)
6923–6931.
[50] M.M. Mullally, H. Meisel, R.J. FitzGerald, Angiotensin-I-converting enzyme inhibitory
activities of gastric and pancreatic proteinase digests of whey proteins, Int. Dairy J. 7
(1997) 299–303.
[51] D. Betancur-Ancona, T. Sosa-Espinoza, J. Ruiz-Ruiz, M. Segura-Campos, L. Chel-
Guerrero, Enzymatic hydrolysis of hard-to-cook bean (Phaseolus vulgaris L.) protein
concentrates and its effects on biological and functional properties, Int. J. Food Sci.
Technol. 49 (2014) 2–8.
[52] F. Scinicariello, M.C. Buser, L. Balluz, K. Gehle, H.E. Murray, H.G. Abadin, R. Attanasio,
Perfluoroalkyl acids, hyperuricemia and gout in adults: analyses of NHANES
2009–2014, Chemosphere 259 (2020), 127446, .
[53] M.H. Oh, K.I. Choe, S.H. Cho, M.S. Jeong, S.C. Myung, S.J. Seo, S.E. Choi, M.W. Lee, Anti-
oxidative and anti-inflammatory effects of flavonoids from the silk of Zea mays Linn,
Asian J. Chem. 25 (8) (2013) 4293–4297.
[54] S. Gao, Y.Y Liu, J.Y. Jiang, X.M. Li, L.X. Zhao, Y. Fu, F. Ye, Encapsulation of thiabenda-
zole in hydroxypropyl-beta-cyclodextrin nanofibers via polymer-free
electrospinning and its characterization, Pest Manag. Sci. 76 (9) (2020) 3264–3272.