Food Chemistry 405 (2023) 134873

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Production, identification and characterization of antioxidant peptides

from potato protein by energy-divergent and gathered ultrasound assisted
enzymatic hydrolysis
Hui Liu a, 1, Hong-Nan Sun a, *, 1, Miao Zhang a, *, Tai-Hua Mu a, *, Nasir Mehmood Khan b
a
Laboratory of Food Chemistry and Nutrition Science, Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Key Laboratory of Agro-
Products Processing, Ministry of Agriculture and Rural Affairs, No. 2 Yuan Ming Yuan West Road, Haidian District, Beijing 100193, China
b
Department of Agriculture, Shaheed Benazir Bhutto University, Sheringal, Upper Dir, Khyber Pakhtunkhwa, Pakistan

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

Keywords: Effects of energy divergent ultrasound (EDU, 30, 40 and 50 W/L) and energy gathered ultrasound (EGU)-assisted
Potato protein hydrolysates enzymatic hydrolysis on production, identification and characterization of antioxidant peptides from potato
Antioxidant peptides protein hydrolysates (PPH) were investigated. EDU and EGU significantly increased degree of hydrolysis and
Amino acid sequence
recovery of PPH compared with conventional (C) enzymatic hydrolysis. PPH by EGU40 showed the highest Fe2+-
Energy-divergent ultrasound
Energy-gathered ultrasound
chelating activity (55.33 µg EDTA/mL), ⋅OH scavenging activity (230.05 µg Vc/mL) and oxygen radical absor­
bance capacity (82.24 µg TE/mL). Peptides with molecular weight <1 and 1–2 kDa of PPH by EGU40 displayed
higher antioxidant activities than those by C and EDU40, which might be contributed to their more hydrophobic,
polar charged and antioxidant amino acids at terminals and/or inside the sequences identified by LC-MS/MS.
Additionally, structure–activity relationship was revealed by peptide conformation and position of some spe­
cific amino acids. Therefore, EGU has great potential in production of functional peptides with enhanced anti­
oxidant activity.

1. Introduction enzymatic hydrolysis and enhance antioxidant activity of protein hy­

In recent decades, plant-derived food proteins have attracted more
and more attention due to their economic, ecological and health benefit
(Yao et al., 2020). Plant proteins can be degraded into bioactive peptides
through enzymatic hydrolysis, e.g. antioxidant peptides (Rahimi et al.,
2022). Antioxidant peptides prepared by enzymatic hydrolysis using
commercial enzymes are generally recognized as safe (GRAS) (Ulug
et al., 2021). Moreover, the application of combinations of different
enzymes can increase the yield and antioxidant activity of protein hy­
drolysates/peptides (Hwang et al., 2016).
As an emerging technology, ultrasound has been widely used in the
food industry and related fields, being highly efficient and convenient
(Yao et al., 2020). Ultrasound is usually classified into two types, energy
divergent ultrasound (EDU) by using an ultrasound bath, and energy
gathered ultrasound (EGU) with an ultrasound probe. The difference
between these two types is the operating conditions and the way of ul­
trasound affecting enzymatic reaction, both of which can promote
drolysates compared with conventional enzymatic hydrolysis (Habin­
shuti et al., 2021).
Potato is the fourth most important food crop after rice, wheat and
corn in the world, and China is the largest potato producer worldwide
with around 78.23 million metric tons in 2020 (FAO, 2020). In the food
industry, potato has become one of the main raw materials for indus­
trialized starch production because of its abundant starch content.
During potato starch manufacturing, a low-value by-product potato fruit
juice (PFJ) is produced, and approximately 3.5 tons of PFJ will be
resulted from the production of each ton of potato starch (Fang et al.,
2011). PFJ contains approximately 1–2 % (w/w) potato protein, which
is of great potential as a food ingredient due to its higher nutritional
quality as compared to proteins from other vegetable and cereal sources,
but has not been fully utilized (Løkra & Strætkvern, 2009). Potato pro­
tein is also considered to be a potential source of antioxidant peptides,
and previous research have reported the functional and conformational
properties (Akbari et al., 2020), possible antioxidant activity (Rahimi

* Corresponding authors.
E-mail addresses: honey0329@163.com (H.-N. Sun), zhangmiao@caas.cn (M. Zhang), mutaihua@126.com (T.-H. Mu).
1
These authors contributed equally: Hui Liu, Hong-Nan Sun.

https://doi.org/10.1016/j.foodchem.2022.134873
Received 25 April 2022; Received in revised form 17 October 2022; Accepted 3 November 2022
Available online 8 November 2022
0308-8146/© 2022 Elsevier Ltd. All rights reserved.
H. Liu et al.
et al., 2022), and potential antioxidant peptide sequences (Habinshuti
et al., 2021) of its hydrolysates. However, to the best of our knowledge,
no study on production, identification and characterization of antioxi­
dant peptides from potato protein by EDU and EGU-assisted enzymatic
hydrolysis has been reported.
In this study, the effects of EDU and EGU-assisted enzymatic hy­
drolysis on production, antioxidant activity and molecular weight (MW)
distribution of potato protein hydrolysates (PPH) were investigated as
compared with non-ultrasonicated conventional (C) samples. Further­
more, antioxidant peptides from PPH by EDU and EGU-assisted enzy­
matic hydrolysis were identified and characterized, of which the
structure–activity dependence was subsequently explored, thus to pro­
vide potential ways to improve antioxidant activity and broaden appli­
cation approach of food proteins.
2. Materials and methods
2.1. Materials
Potato protein (72.10 % protein, 6.36 % moisture, 4.91 % crude
fiber, 3.49 % ash, 1.40 % reducing sugar, and 0.94 % fat) was provided
by Zhuanglang Hongda Starch Processing Co., ltd. (Gansu, China).
Alcalase 2.4 l® (nominal activity of 2.4 Anson unit per gram (AU/g)),
Flavourzyme 500 MG® (nominal activity of 500 Leucine Aminopepti­
dase units per gram (LAPU/g)) and Neutrase 0.8 l® (nominal activity of
0.8 AU/g) were purchased from Novozymes (Bagsvaerd, Denmark). The
2, 20-azobis (2-amidino propane) dihydrochloride (AAPH) was pur­
chased from APExBIO Technology LLC (Shanghai, China). 6-hydroxy-2,
5, 7, 8-tetramethlchroman-2-carboxylic acid (Trolox, 98 %) was ob­
tained from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Other reagents
were all analytical grades.
2.2. Energy-divergent (EDU) and gathered ultrasound (EGU)-assisted
enzymatic hydrolysis of potato protein
The EDU and EGU-assisted enzymatic hydrolysis of potato protein by
the combination of Alcalase, Neutrase and Flavourzyme were performed
according to the method of Habinshuti et al., (2021) with modifications.
Briefly, 3 g of potato protein was dissolved in 100 mL of distilled water
and then hydrolyzed with successive enzymatic hydrolysis at 4 % (w/w)
enzyme to substrate ratio at 50 ◦ C firstly by Alcalase at pH 8.0 for 1 h,
followed by Neutrase at pH 6.0 for 1 h, and finally by Flavourzyme at pH
7.0 for 1 h respectively. For EDU-assisted enzymatic hydrolysis, the re­
action was performed by using an ultrasonic bath (SK8210HP, Shanghai
Kudos Ultrasonic Instrument Co. ltd., Shanghai, China) at a power
density of 30, 40 and 50 W/L respectively. For EGU-assisted enzymatic
hydrolysis, the reaction was performed by using an ultrasound probe
(Scientz-IID, Ningbo Scientz Biotechnology Co., ltd, Zhejiang, China)
with the amplitude of 2 %, off/on time of 5 s, and a power density of 30,
40 and 50 W/L respectively. The non-ultrasonicated conventional (C)
enzymatic hydrolysis was performed by using the shaking incubator
(DSHZ-300A, Taicang Experimental Equipment Factory, Jiangsu, China)
at a rotation speed of 80 rpm. After 3 h of hydrolysis, each hydrolysate
was placed in the boiling water for 15 min, transferred to ice water
immediately for cooling down, and centrifuged at 10,000 g for 20 min at
4 ◦ C, then the supernatant was collected and stored at − 40 ◦ C for freeze-
drying.
2.3. Degree of hydrolysis (DH) and recovery
DH of PPH was conducted by o-phthal-dialdehyde (OPA) reaction
according to the method by Nielsen et al. (2001).
DH(%) = (h/htot ) × 100
where htot means peptide bonds number of one unit protein equivalent,
2
Food Chemistry 405 (2023) 134873
being approximately 8 g equivalent/kg protein.
Recovery of PPH is the ratio of protein content of the lyophilized PPH
to the protein content of potato protein powder used for enzymatic
hydrolysis (Udenigwe & Aluko, 2010). Protein content in the superna­
tant was determined by the method described by Markwell et al.,
(1978).
2.4. Hydroxyl radical (•OH) scavenging activity assay
Hydroxyl radical (•OH) scavenging activity assay was based on the
method of Zhang & Mu (2016) with modifications. A portion of 0.2 mL
of PPH solution (1 mg protein/mL) was mixed well with 0.1 mL of 10
mM FeSO4, 0.5 mL of 10 mM α-deoxyribose, 0.9 mL of phosphate buffer
(pH 7.4), and 0.1 mL of 10 mM disodium EDTA by using a vortex mixer
(SCI-vS Scilogex, MX-RD-E, USA) for 7 s. After that, 0.2 mL of 10 mM
H2O2 was added, mixing sufficiently, and incubated at 37 ◦ C for 1 h in
darkness. Then 1.0 mL of trichloroacetic acid (TCA, 2.8 %, w/v) was
added, and mixed sufficiently. Subsequently, 1.0 mL of thiobarbituric
acid (TBA, 1.0 %, w/v) was added into the solution, kept in a water bath
at 100 ◦ C for 15 min, and then cooled down to room temperature. The
absorbance of the mixture at 532 nm was determined using a spectro­
photometer (U-3010, Tokyo Hitachi Co., ltd., Tokyo, Japan), and •OH
scavenging activity was presented with μg Vc (ascorbic acid)/mL.
2.5. Fe2+-chelating activity assay
Fe2+-chelating activity assay was referred to Zhang and Mu (2016)
with modifications. Briefly, 900 µL of 1 mg protein/mL PPH solution
were mixed with 90 µL of FeSO4 (2 mM) and 3630 µL of distilled water,
and maintained at 25 ◦ C for 30 min. Afterwards 90 µL of ferrozine (5
mM) was added, mixed sufficiently, and A562 nm was instantly detected
by a spectrophotometer (U-3010, Tokyo Hitachi Co., ltd., Tokyo, Japan).
Fe2+-chelating activity was presented with μg EDTA/mL.
2.6. Oxygen radical absorbance capacity (ORAC) assay
ORAC assay of PPH was based on the method by Habinshuti et al.,
(2021) with modifications. All solution was prepared with phosphate
buffer (75 mM, pH 7.4). Firstly, 20 μL of 1 mg protein/mL PPH solution,
20 μL of sodium fluorescein (63 nM) and 20 μL phosphate buffer were
added to a microplate, mixed gently, and stored at 37 ◦ C for 10 min in
the dark. After that, AAPH solution (140 μL, 18.28 mM) was instantly
added, and the fluorescence was detected by a Fluoroskan Ascent
microplate reader (Thermo Electron Corporation, Vantaa, Finland)
every 2 min under 485-P excitation and 520-P emission filters for a total
of 60 detections. The ORAC value was presented with μg TE (Trolox
equivalent)/mL.
2.7. Molecular weight (MW) distribution
MW distribution was referred to the method of Habinshuti et al.,
(2021) with modifications. A portion of 10 mg of PPH was dissolved in 1
mL of 50 mM phosphate buffer (0.15 M sodium chloride, pH 7.0), and
was filtered through a 0.22 µm pore size hydrophilic Polyethersulfone
membrane. Each PPH solution was diluted to a protein concentration of
1 mg/mL, and detected on RP-HPLC (Shimadzu LC-20A, Shimadzu
Corporation, Kyoto, Japan) at 215 nm with a Superdex 30 Increase 10/
300 GL column (10 × 300 mm, GE healthcare Bio-Sciences AB, Uppsala,
Sweden). The injection volume was 20 µL, and the column temperature
was 25 ◦ C. Eluent was 50 mM phosphate buffer with 0.15 M sodium
chloride (pH 7.0), and the flow rate was 0.5 mL/min for 70 min. The
absorbance of the effluent was monitored at 215 nm. Cytochrome c
(12,384 Da), aprotinin (6,512 Da), vitamin B12 (1,355 Da) and glycine
(75 Da) from Sigma-Aldrich, Inc. (St. Louis, MO, USA) were used as
standards to build MW calibration curve based on the average retention
time.
H. Liu et al. Food Chemistry 405 (2023) 134873

2.8. Peptide fractionation by AKTA pure system
Fractionation of PPH was performed with the method of Falade et al.
(2021) with modifications by using AKTA™ PURE M1 (Varian Prostar,
Agilent Technologies, Santa Clara, CA, USA) on a Superdex 30 Increase
10/300 GL column (10 × 300 mm). Each 10 mg protein/mL of PPH
solution was subjected to fractionation with eluent of 50 mM phosphate
buffer with 0.15 M sodium chloride (pH 7.0) at 0.5 mL/min flow rate.
Injection volume was 500 µL, and column temperature was 25 ◦ C.
Elution peaks were detected at 215 nm, and fractions with MW > 7, 5–7,
3–5, 1–2 and <1 kDa were collected. Difference MW fractions at 100 µg
protein/mL were further screened for ORAC, and fractions with superior
ORAC were selected for further separation by semi preparative reverse-
phase high performance liquid chromatography (RP-HPLC).
2.9. Peptide separation by semi preparative RP-HPLC
Peptide separation was performed by using semi preparative RP-
HPLC (Agilent Technologies, Santa Clara, CA, USA) on a Varian C18
column (21.2 × 150 mm). The eluent was trifluoroacetic acid (TFA, 0.1
%, v/v) (mobile phase A) and acetonitrile (mobile phase B). The injec­
tion volume was 1 mL, and the column temperature was 25 ◦ C. Gradient
elution was performed at 0–30 min with linear gradient 5–20 % mobile
phase B, then 30–60 min with isocratic gradient 20 % mobile phase B,
and the flow rate was 10 mL/min. Each fraction (4 mL) was collected,
lyophilized, and mixed with 200 μL of 75 mM phosphate buffer (pH 7.4)
for ORAC evaluation. Peptide fractions with the highest ORAC value
were selected for further identification by liquid chromatography-
tandem mass spectrometry (LC-MS/MS).
2.10. Identification and characterization of identified peptides
Amino acid sequences of the selected peptide fractions were identi­
fied by LC-MS/MS coupled with an Eksigent Nano LC (Eksigent Tech­
nologies, Dublin, CA, USA) and Thermo LTQ linear ion trap mass
spectrometer (Thermo Fisher, San Jose, CA, USA) on an LC Packings
PepMap300 C18 column (particle size 5 μm; pore size 300 Å; 75 μm ×
150 mm, Dionex, Sunnyvale, CA, USA). The eluent was consisted of 2 %
acetonitrile, 0.1 % formic acid (mobile phase A) and 80 % acetonitrile,
0.1 % formic acid (mobile phase B). Gradient elution was performed at
0–85 min with linear gradient 5 %–50 % mobile phase B, then 85–95
min with linear gradient 50 %–95 % mobile phase B, finally 95–125 min
with isocratic gradient 95 % mobile phase B at a flow rate of 300 nL/
min. The MS/MS data were utilized to evaluate the sequences of eluted
peptides with a spray voltage of 2.2 kV, a scan range of m/z 400–2000, a
data acquisition time of 110 min, a capillary temperature of 200 ◦ C, and
of DH with recovery of PPH was investigated by Pearson correlation
analysis. The differences were considered significant at P < 0.05.
3. Results and discussion
3.1. DH and recovery of PPH as influenced by EDU and EGU-assisted
enzymatic hydrolysis
DH and recovery of PPH as influenced by EDU and EGU-assisted
enzymatic hydrolysis were presented in Table 1. DH of PPH by EDU
assisted hydrolysis were 18.84 %-26.38 % with power density increasing
from 30 to 50 W/L, while DH of PPH by EGU were increased from 26.29
% to 37.52 %, which were significantly higher than that by non-
ultrasonicated conventional hydrolysis (9.19 %, P < 0.05, Table 1).
Obviously, two types of ultrasounds assisted enzymatic hydrolysis
significantly increased DH of PPH (P < 0.05, Table 1). Ultrasound could
produce transient cavitation effects, which could crack polymers
randomly by severe shearing force (X. Yang et al., 2017). Ultrasound
could also lead to the generated of free radicals by the dissolution of
water molecules, which further changed the structure of the substrate,
and resulted in the increase in DH during enzymatic hydrolysis (Weiss
et al., 2011). In addition, under the same power density of 30, 40 or 50
W/L, DH of PPH by EGU was significantly higher than that by EDU (P <
0.05, Table 1). Corn protein hydrolysates by EGU also showed higher DH
than that by EDU (Huang et al., 2015). The possible reason was that EGU
could produce stronger cavitation and turbulent effects, leading to the
exposure of more charged groups and enhanced protein-water in­
teractions (Liu et al., 2017). However, DH of PPH by EGU50 (34.37 %)
was lower than that by EGU40 (37.52 %, P < 0.05, Table 1). This might
be due to the change in the conformation of the protein during the
higher energy ultrasound process, and much more hidden hydrophobic
groups were exposed to protein surface that reduced available peptide
bonds and hindered the hydrolysis process (Kangsanant et al., 2014).
The decrease in DH was also observed in tilapia (Oreochromis niloticus)
hydrolysate by ultrasound-assisted Flavourzyme hydrolysis at higher
power (Kangsanant et al., 2014).
In the case of protein recovery, compared to PPH by non-
ultrasonicated conventional enzymatic hydrolysis (21.97 %), PPH by
Table 1
Effect of different ultrasonic-assisted enzymatic hydrolysis on DH, recovery and
antioxidant activity of potato protein hydrolysates.
Treatment DH Recovery Fe2 ⋅OH ORAC
(%) (%) ＋
chelating scavenging (μg TE/
activity (μg activity (μg mL)
EDTA/mL) Vc/mL)

a normalised collision energy of 35 %. Finally, the MS/MS data were C 9.19 ± 25.26 ± 44.56 ± 94.98 ± 65.45 ±
analyzed by Mascot (version 2.1.0, Matrix Sciences, London, UK). Pep­ 0.16e 2.03d 0.25f 2.23d 5.49d
EDU30 18.44 31.96 ± 53.13 ± 216.41 ± 72.77 ±
tide sequences were compared to the potato protein sequences from the
± 2.41cd 0.19d 2.59b 7.38c
National center for Biotechnology Information (NCBI, Bethesda USA) 0.69d
database. EDU40 25.08 40.53 ± 54.66 ± 225.76 ± 76.71 ±
± 0.19c 2.97c 0.02b 2.12ab 12.65bc
2.11. Peptide conformation EDU50 26.38 41.59 ± 54.43 ± 225.05 ± 74.06 ±
± 0.72c 3.61c 0.11b 1.09ab 5.13bc
EGU30 26.29 63.98 ± 53.89 ± 200.31 ± 79.89 ±
Conformational structure of the peptides was carried out by PEP- ± 0.30c 6.40ab 0.06c 1.82c 7.41b
FOLD3 according to Lamiable et al. (2016), which could be used for EGU40 37.52 69.09 ± 55.33 ± 230.05 ± 82.24 ±
faster de novo linear peptide structure. An interactive 3D visualization of ± 5.25a 0.01a 2.97ab 3.63a
0.29a
the peptides with the best models was presented using the JavaScript
EGU50 34.37 55.94 ± 51.34 ± 236.16 ± 78.33 ±
viewer (http://biasmv.github.io/pv). ± 6.56b 0.04e 2.47a 8.34b
1.10b
2.12. Statistical analysis
C: non-ultrasonicated conventional hydrolysis; EDU30, EDU40 and EDU50:
energy divergent ultrasound assisted enzymatic hydrolysis with power density
Each experiment was carried out in triplicate, and the data were of 30, 40 and 50 W/L respectively; EGU30, EGU40 and EGU50: energy gather
expressed as means ± SD. Statistical analysis was performed using one- ultrasound assisted enzymatic hydrolysis with power density of 30, 40 and 50
way ANOVA followed by a Duncan multiple-comparison test with SPSS W/L. Values followed by different superscript lowercase letters in the same
27.0 software (IBM Corporation, NY, USA). The correlation coefficient column mean statistically significant differences (p < 0.05).

3
H. Liu et al.
EDU and EGU-assisted enzymatic hydrolysis showed significantly higher
recovery (P < 0.05, Table 1). For EDU, recovery of PPH was 31.96 %,
40.53 % and 41.59 % for EDU30, EDU40 and EDU50 respectively (P >
0.05, Table 1). While for EGU, recovery of PPH was 63.98 %, 69.09 %
and 55.94 % for EGU30, EGU40 and EGU50 respectively (P < 0.05,
Table 1). Obviously, recovery of PPH by EGU40 was the highest (P <
0.05, Table 1), followed by EGU30, and then EGU50. The excessive in­
crease in ultrasound power led to greater cavitation, mechanical and
thermal consequences, which might affect the molecular conformation
of the protease, thereby reducing the hydrolysis efficiency and being
detrimental to yield enhancement (Yu et al., 2012). In addition, the
recovery of PPH showed a strongly positive correlation with DH in this
study (r = 0.8710, P < 0.05), which was also observed in the recovery
and DH of protein hydrolysate obtained from Chinese sturgeon (Noman
et al., 2020).
Fig. 1. RP-HPLC chromatograms (a, b and c) and molecular weight distribution (d) of PPH by EDU and EGU at the power density of 30, 40 and 50 W/L respectively.
C, non-ultrasonicated conventional enzymatic hydrolysis; EDU, energy divergent ultrasound; EGU, energy gather ultrasound.
4
Food Chemistry 405 (2023) 134873
3.2. Antioxidant activities of PPH as influenced by EDU and EGU-assisted
enzymatic hydrolysis
Antioxidant activities of PPH as influenced by EDU and EGU-assisted
enzymatic hydrolysis were shown in Table 1. Compared with PPH by
EDU and EGU assisted enzymatic hydrolysis, PPH by non-ultrasonicated
conventional enzymatic hydrolysis had the lowest Fe2+-chelating ac­
tivity (44.56 µg EDTA/mL), ⋅OH scavenging activity (94.98 µg Vc/mL)
and ORAC value (65.45 µg TE/mL) (P < 0.05). Both EDU and EGU-
assisted enzymatic hydrolysis enhanced Fe2+-chelating activity, ⋅OH
scavenging activity and ORAC values of PPH significantly (P < 0.05,
Table 1). Among all hydrolysates, PPH by EGU40 showed the highest
Fe2+-chelating activity (55.33 µg EDTA/mL), followed by EDU40
(54.66 µg EDTA/mL) (P < 0.05). PPH by EGU50 presented the highest
⋅OH scavenging activity (236.16 µg Vc/mL), followed by EGU40
(230.05 µg Vc/mL), but showed no significant difference between each
other (P > 0.05). Moreover, ORAC value of PPH by EGU40 was the
H. Liu et al.
Fig. 1. (continued).
highest (82.24 μg TE/mL), successively followed by EGU30, EGU50 and
EDU40 (P < 0.05, Table 1). Higher antioxidant activity of the hydroly­
sates by ultrasound-assisted enzymatic hydrolysis might be due to that
ultrasound could alter the secondary and tertiary structures of proteins
through the disruption of hydrogen bonds and hydrophobic interactions,
inducing molecular unfolding and/or protein defragmentation (Mag­
alhães et al., 2022). These changes could lead to the exposure of sulf­
hydryl and hydrophobic groups of proteins, facilitating the enzyme to
access polypeptide chain regions that susceptible to hydrolysis, conse­
quently improving the efficiency of enzymatic hydrolysis and release of
antioxidant peptides (F. Li & Tang, 2021).
Furthermore, it was worth noting that compared to EDU, PPH by
EGU showed higher antioxidant activities (Table 1). EDU and EGU were
also used in the preparation of sweet potato protein hydrolysates, and
the hydrolysates obtained by EGU showed stronger antioxidant activity
than that by EDU (Habinshuti et al., 2021). The possible reason might be
due to that EGU brought out more uniform cavitation, thus could reduce
the decomposition of antioxidant substances caused by excessive cavi­
tation (Walters et al., 2020). Moreover, PPH by EGU40 had higher Fe2+-
chelating activity and ORAC than that by EGU50 (Table 1). It might be
due to that high ultrasound power would destroy the enzyme structure,
thereby resulting in the inactivation or reduction of enzyme activity and
making it unsuitable for the subsequent enzymatic hydrolysis of the
substrate (Yang et al., 2021).
3.3. MW distribution of PPH as influenced by EDU and EGU-assisted
enzymatic hydrolysis
MW distribution of PPH as influenced by EDU and EGU-assisted
enzymatic hydrolysis was shown in Fig. 1 a, b, c and d. Compared
with non-ultrasonicated conventional enzymatic hydrolysis, PPH by
EDU and EGU presented higher proportion of fractions with lower MW
2–3, 1–2 and <1 kDa (Fig. 1 a, b and c). For non-ultrasonicated con­
ventional enzymatic hydrolysis, the percentage of MW > 7, 5–7, 3–5,
2–3, 1–2 and <1 kDa fractions in PPH was 4.15 %, 3.93 %, 13.37 %,
5
Food Chemistry 405 (2023) 134873
21.43 %, 34.68 % and 22.45 % respectively (P < 0.05, Fig. 1 d). The
proportion of MW 1–2 and <1 kDa fractions in PPH by EDU40 and
EGU40 was significantly higher than that by non-ultrasonicated con­
ventional enzymatic hydrolysis, which were 38.31 % and 23.54 % for
EDU40, and 38.82 % and 23.85 % for EGU40 respectively (P < 0.05,
Fig. 1 d). Obviously, PPH by EDU40 (62.68 %) and EGU40 (61.85 %)
contained more fractions with MW < 2 kDa than that by conventional
enzymatic hydrolysis (57.13 %, P < 0.05, Fig. 1 d). Ultrasound also
showed a promotion on the production of lower MW fractions in sweet
potato protein hydrolysates, which might be due to the higher DH
during ultrasound-assisted enzymatic hydrolysis (Habinshuti et al.,
2021).
3.4. Effect of MW distribution on antioxidant activities of different PPH
ORAC values of different MW fractions from PPH as influenced by
EDU and EGU-assisted enzymatic hydrolysis were shown in Fig. 2. MW
1–2 kDa fractions of PPH by C, EDU40 and EGU40 had higher ORAC
values as compared to other MW fractions, which were 59.65, 70.83 and
82.47 µg TE/mL respectively (P > 0.05, Fig. 2), followed by MW < 1 kDa
fraction of PPH by C, EDU40 and EGU40, of which ORAC values were
55.99, 63.04 and 66.26 µg TE/mL respectively (P > 0.05, Fig. 2). In
addition, MW 2–3, 3–5 and 5–7 kDa fractions of PPH by C, EDU40 and
EGU40 showed much higher ORAC values as compared to MW > 7 kDa
fractions (P < 0.05, Fig. 2). Higher antioxidant activity of MW 1–2 and
<1 kDa fractions might be due to the production of more low MW
peptides with stronger antioxidant capacities (Yu et al., 2012). Some
other studies also indicated that peptides with MW 1–3 kDa showed
higher antioxidant activity (Zhou et al., 2013; Zou et al., 2016), of which
the higher antioxidant activity was related to the composition and
sequence of amino acids, as well as the position of hydrophobic amino
acids (Ketnawa et al., 2018; Wu et al., 2020).
H. Liu et al. Food Chemistry 405 (2023) 134873

100 3.5. Separation and antioxidant activities of MW 1–2 and < 1 kDa
Aa peptide fractions
C
80 ABa Ab EDU40 MW 1–2 and <1 kDa fractions of PPH by C, EDU40 and EGU40 were
EGU40 selected due to their high antioxidant activities, and separated into
ABa
ABa
ABa different fractions based on peak recognition by semi preparative RP-
Ac
Ba BCa HPLC (Table 2). As shown in Table 2, 10, 15 and 14 peak fractions
ORAC (μgTE/mL)

60 Aa Aa Ba Aa Aa
Ba
were separated from MW < 1 kDa peptides of PPH by C, EDU40 and
EGU40 respectively, while 23, 23 and 24 peak fractions were obtained
40 from MW 1–2 kDa fractions of PPH by C, EDU40 and EGU40 respec­
Ca tively. In the case of PPH by C, fraction No. 3 in MW < 1 kDa peptides
Cb showed the highest ORAC value, which was 55.50 µg TE/mL, while
20
Bc fraction No. 17 of MW 1–2 kDa peptides showed the highest ORAC
value, which was 53.72 µg TE/mL (P > 0.05, Table 2). For PPH by
EDU40, fraction No. 8 in MW < 1 kDa and fraction No. 8 in MW 1–2 kDa
peptides showed the highest ORAC values, which were 70.89 and 63.45
0
<1 1-2 2-3 3-5 5-7 >7 µg TE/mL respectively (P > 0.05, Table 2). For PPH by EGU40, fraction
Molecular Weight (kDa) No. 4 in MW < 1 kDa and fraction No. 9 in MW 1–2 kDa peptides showed
the highest ORAC values, which were 79.07 and 78.48 µg TE/mL
Fig. 2. Oxygen radical absorbance capacity (ORAC) of PPH by EDU40 and respectively (P > 0.05, Table 2). Obviously, peak fractions in MW < 1
EGU40 with different molecular weight. C, non-ultrasonicated conventional and 1–2 kDa from PPH by EGU40 showed higher ORAC values than
enzymatic hydrolysis; EDU40, energy divergent ultrasound at 40 W/L; EGU40, those by C and EDU40 (P < 0.05, Table 2). During chromatographic
energy gather ultrasound at 40 W/L. fractionation by RP-HPLC, peptide fractions with stronger polarity were

Table 2
ORAC of different peaks from <1 and 1–2 kDa fractions in potato protein hydrolysates as influenced by different ultrasonic-assisted enzymatic hydrolysis.
MW (kDa) Fraction C EDU40 EGU40
No.
Time (min) ORAC Time (min) ORAC Time (min) ORAC
(µg TE/mL) (µg TE/mL) (µg TE/mL)

<1 1 9.67–11.34 44.79 ± 5.07bc 9.83–10.75 14.14 ± 1.66e 9.67–11.00 71.36 ± 3.52b
2 11.44–11.67 34.35 ± 0.27d 10.85–11.08 26.05 ± 2.22c 11.10–12.60 64.61 ± 0.41c
3 11.77–12.17 55.50 ± 4.24a 11.18–12.16 25.02 ± 3.13cd 12.70–13.17 60.24 ± 0.13cd
4 12.27–12.75 36.55 ± 0.24d 13.91–14.66 43.10 ± 4.50b 13.27–13.67 79.07 ± 2.28a
5 12.85–13.92 25.36 ± 4.19e 15.66–16.65 50.70 ± 5.14b 13.77–14.33 43.69 ± 2.98e
6 14.17–14.50 2.69 ± 0.07g 29.58–30.16 24.16 ± 6.96cd 14.43–14.75 19.74 ± 1.21fg
7 14.60–16.25 14.04 ± 1.50f 30.26–31.66 19.07 ± 7.82cde 14.85–15.75 57.23 ± 1.52d
8 16.35–18.50 40.26 ± 1.85cd 32.16–33.00 70.89 ± 8.07a 16.50–18.75 23.58 ± 0.57f
9 30.67–31.84 48.37 ± 2.49b 33.10–34.00 67.32 ± 9.84a 18.85–21.10 10.60 ± 0.47h
10 31.94–33.34 37.43 ± 2.59d 34.10–35.00 22.23 ± 10.65cde 23.30–26.08 17.06 ± 1.86g
11 – – 36.25–36.83 13.78 ± 11.80e 36.42–37.00 21.46 ± 1.33fg
12 – – 38.93–40.41 22.29 ± 12.18cde 37.10–38.15 38.81 ± 2.49e
13 – – 42.51–43.91 16.66 ± 13.11de 38.27–39.25 71.39 ± 6.05b
14 – – 44.01–44.91 20.49 ± 14.60cde 46.20–51.15 7.95 ± 0.19h
15 – – 45.01–45.66 5.41 ± 15.23f – –
1–2 1 10.06–11.81 8.12 ± 0.32kl 9.65–11.38 13.54 ± 0.20m 10.31–12.39 10.97 ± 1.28ij
2 13.89–14.23 44.54 ± 1.50b 11.50–13.00 57.51 ± 0.94b 14.65–15.73 48.59 ± 2.37c
3 14.33–14.56 38.90 ± 0.96c 13.10–14.80 49.20 ± 3.08cd 16.40–18.31 24.47 ± 3.99 h
4 14.66–15.98 34.48 ± 1.34d 15.00–16.10 26.48 ± 0.78hi 19.73–20.56 24.92 ± 4.03h
5 19.39–19.64 28.96 ± 5.83ef 17.01–18.75 23.30 ± 0.50ij 22.56–23.20 66.59 ± 5.48b
6 19.81–19.98 3.57 ± 0.17l 19.20–20.65 31.61 ± 0.58fg 23.64–24.04 12.51 ± 6.42i
7 20.08–20.31 3.60 ± 0.14l 20.75–21.83 27.12 ± 0.93hi 24.14–24.90 34.26 ± 7.76defg
8 21.43–21.89 13.20 ± 1.50ij 21.93–22.50 63.45 ± 1.73a 25.39–26.39 34.95 ± 8.79defg
9 21.99–22.64 10.87 ± 0.43jk 23.64–26.20 18.11 ± 0.13kl 27.23–27.81 78.48 ± 9.22a
10 25.14–26.23 6.80 ± 0.03kl 24.60–26.60 39.79 ± 3.31e 30.06–30.84 28.19 ± 10.3fg
11 29.06–29.73 32.79 ± 2.42de 27.10–28.85 14.55 ± 0.69lm 30.94–31.48 28.67 ± 11.97fgh
12 30.23–30.79 15.43 ± 0.86ij 30.10–32.00 28.67 ± 3.09gh 31.58–33.48 13.76 ± 12.1i
13 30.89–31.73 44.31 ± 2.60b 32.18–34.35 9.12 ± 0.12n 33.65–34.31 5.52 ± 13.9j
14 32.06–32.64 13.73 ± 0.26ij 34.45–35.48 4.08 ± 0.34o 34.48–35.31 39.05 ± 14.59d
15 34.06–34.64 5.15 ± 1.96l 36.20–37.85 33.76 ± 0.97f 36.98–37.48 29.07 ± 15.1fgh
16 34.89–35.81 17.87 ± 2.07hi 38.00–39.45 20.30 ± 3.05jk 37.58–38.73 75.75 ± 16.51a
17 35.91–36.56 53.72 ± 0.76a 39.55–40.85 8.75 ± 0.07n 38.83–39.23 74.91 ± 17.24a
18 36.98–38.38 24.64 ± 2.04g 41.12–41.90 16.05 ± 0.18lm 39.56–39.89 38.76 ± 18.17de
19 41.81–42.10 6.36 ± 2.01kl 42.00–44.20 45.36 ± 2.29d 39.99–40.81 52.71 ± 19.21c
20 42.30–42.61 25.94 ± 0.10fg 44.50–45.03 52.12 ± 5.13c 41.14–41.64 35.58 ± 20.94def
21 45.23–46.80 21.85 ± 1.61gh 47.20–49.65 26.92 ± 0.46hi 41.74–42.98 31.21 ± 21.54efg
22 51.90–52.50 32.55 ± 0.72de 49.75–50.85 33.59 ± 1.34f 43.08–44.15 55.53 ± 22.61c
23 55.39–56.06 15.56 ± 1.16i 50.00–51.5 5.55 ± 1.31no 44.25–45.56 34.74 ± 23.38defg
24 – – – – 47.81–48.53 50.83 ± 24.19c

C: non-ultrasonicated conventional hydrolysis; EDU40: energy divergent ultrasound assisted enzymatic hydrolysis with power density of 40 W/L; EGU40: energy
gather ultrasound assisted enzymatic hydrolysis with power density of 40 W/L. Values followed by different superscript lowercase letters in the same column mean
statistically significant differences (P < 0.05).

6
H. Liu et al. Food Chemistry 405 (2023) 134873

eluted first, while those with less polarity came out later (Jiang et al., Table 3
2019). In this study, fractions No. 3 and 4 in MW < 1 kDa peptides of Peptide sequences from selected fractions identified by LC-MS/MS.
PPH by C and EGU40 were eluted earlier, fractions No. 8 (MW < 1 kDa, Fraction Peptide MW Matched sequences*
EDU40), No. 8 (MW 1–2 kDa, EDU40) and No. 9 (MW 1–2 kDa, EDU40) no. (Da)
were eluted out in the middle, while fraction No. 17 (MW 1–2 kDa, C) 3 VKDNPLDVS 985.51 f208 - patatin
was eluted later (Table 2), suggesting their different polarity. Antioxi­ 216
dant capacity of peptides was not only due to the available polar and (<1 kDa, C) SIDGGG 504.22 f33 - patatin
ionizable groups after enzymatic cleavage, but also largely depended on 38
SIDGGGIK 745.4 f33 - patatin
their molecular size, hydrophobicity and amino acid sequences (Li et al., 40
2021; Liu et al., 2010). Thus, further identification and characterization SIDGGGIKG 802.42 f33 - patatin
of each peptide fraction would help to understand the potential cause of 41
their high antioxidant activity more clearly. IGGTS 433.22 f73 - patatin
77
TSNGDKYE 912.38 f203 - patatin
3.6. Identification and characterization of peptide sequences 210
GDKYE 610.26 f206 - patatin
All identified peptides belonged to potato patatin, Cys protease in­ 210
DGAVA 431.20 f215 - patatin
hibitor, aspartic proteinase inhibitor, kunitz-type protease inhibitor,
219
serine protease inhibitor or proteinase inhibitor I (Table 3). Totally 23, TVGDPA 558.27 f220 - patatin
17 and 40 peptide sequences in selected fractions from PPH with MW < 225
1 kDa by C, EDU and EGU were identified, whereas 6, 18 and 66 peptide FDKTY 672.31 f266 - patatin
sequences were found in selected fractions from PPH with MW 1–2 kDa 270
FDKTYT 773.36 f266 - patatin
by C, EDU and EGU respectively (Table 3).
271
High proportion of hydrophobic amino acids of peptides was re­ VQVGE 530.27 f343 - patatin
ported to contribute to their higher antioxidant activity (Wattanasiri­ 347
tham et al., 2016; Zhu et al., 2020). Bulky hydrophobic residues at C- VSKDSPE 760.36 f354 - patatin
360
and N-terminus were also common in free radical scavenging activities
DQDGNPL 757.32 f8 - 14 Cys protease
(Zou et al., 2016). In selected fractions from PPH with MW < 1 kDa inhibitor
peptides by C, EDU and EGU, 20, 15 and 38 peptide sequences con­ IGERY 636.32 f16 - Cys protease
taining hydrophobic amino acids were detected respectively (Table 3). 20 inhibitor
Among them, 1, 4 and 9 peptide sequences from PPH with MW < 1 kDa GGKVGNE 659.32 f111 - Cys protease
117 inhibitor
peptides by C, EDU and EGU had hydrophobic amino acids located at N-
VTVDDDK 772.40 f164 - Cys protease
and/or C-terminus (marked in bold, Table 3). While for MW 1–2 kDa 170 inhibitor
peptides by C, EDU and EGU, 6, 17 and 66 peptide sequences contained NSDVGPS 674.29 f84 - aspartic
hydrophobic amino acids respectively, and 1, 3 and 8 peptide sequences 90 proteinase
had them at N- and/or C-terminus (showed in bold, Table 3). The pro­ inhibitor
SDVGPS 560.24 f85 - aspartic
portion of these hydrophobic peptides in total peptides was 89.66 %, 90 proteinase
91.43 % and 98.11 % for C, EDU and EGU respectively. Obviously, PPH inhibitor
by EGU produced more hydrophobic peptides, which presented higher DVGPS 473.21 f86 - aspartic
antioxidant capacity as shown in Table 1. In addition, the peptides of 90 proteinase
inhibitor
MW of 1–2 fractions from PPH by EGU had more hydrophobic sequences
NSDVGPS 674.29 f88 - kunitz-type
than MW < 1 kDa, which was in concert with its significantly higher 94 protease
antioxidant activities (Fig. 2). inhibitor
Polar charged amino acids at the C-terminus also contributed to the NSDVGPS 674.29 f84 - serine protease
increased antioxidant activity of the peptides (Ketnawa et al., 2018). 90 inhibitor
SDVGPS 560.24 f85 - serine protease
Peptides with aspartic (D) and glutamic acid (E) at the C-terminus could 90 inhibitor
scavenge hydroxyl radicals, and chelate metal ions by binding charged SIDGGGIK 745.40 f33 - patatin
ions to metal (Pan et al., 2016; Udenigwe & Aluko, 2010). There were 7 40
(C, <1 kDa), 3 (C, 1–2 kDa), 5 (EDU, <1 kDa), 10 (EDU, 1–2 kDa), 14 NNRPF 646.32 f92 - patatin
96
(EGU, <1 kDa), and 25 (EGU, 1–2 kDa) peptides with C-terminal polar/
8 KDIVPF 717.41 f100 - patatin
charged amino acids among identified peptide sequences respectively 105
(Table 3). Among them, 1, 3, 4, 2, 2 and 19 peptides sequences with C- (<1 kDa, HGPHIF 706.36 f109 - patatin
terminal basic positively charged amino acids were obtained from each EDU40) 114
fraction mentioned above respectively (marked in bold, Table 3). IFGPM 563.28 f120 - patatin
124
Obviously, selected fractions from MW 1–2 kDa peptides in PPH by EGU SFDIK 608.32 f154 - patatin
had the most abundant peptides with C-terminal basic charged amino 158
acids, which also contributed to its significantly higher ORAC value TNKPVIF 817.47 f159 - patatin
(Fig. 2). In addition, 6, 0, 1, 6, 12 and 13 peptides derived from C (<1 165
AQVDPKF 803.42 f236 - patatin
kDa), C (1–2 kDa), EDU (<1 kDa), EDU (1–2 kDa), EGU (<1 kDa), and
242
EGU (1–2 kDa) had acidic negatively charged amino acids at C-terminal, HSQNNYL 874.39 f311 - patatin
such as aspartic (D) and glutamic acid (E) (underlined, Table 3). Pep­ 317
tides with MW < 2 kDa of EGU (22.64 %) had the abundant D and E VSKDSPE 760.36 f354 - patatin
amino acids than C (17.24 %) and EDU (20 %), which indicated its 360
ALKRF 633.40 f365 - patatin
higher Fe2+-chelating activity and ⋅OH scavenging activity (Table 1). 369
The presence of aromatic residues (Y, W, and F), imidazolyl (H), and SDYGDVVR 909.42
sulfur-containing amino acids (C and M) in peptides could also help to (continued on next page)
scavenge free radicals through direct electron transfer (Nimalaratne

7
H. Liu et al. Food Chemistry 405 (2023) 134873

Table 3 (continued ) Table 3 (continued )

Fraction Peptide MW Matched sequences* Fraction Peptide MW Matched sequences*
no. (Da) no. (Da)

f70 - Cys protease NSDVGPS 674.29 f84 - aspartic

77 inhibitor 90 proteinase
NDIFK 635.33 f118 - Cys protease inhibitor
122 inhibitor FENEL 650.29 f104 - aspartic
VKDNPLDVS 985.51 f204 - aspartic 108 proteinase
212 proteinase inhibitor
inhibitor DLPSE 559.25 f29 - kunitz-type
GTPVRFIPL 998.59 f95 - kunitz-type 33 protease
103 protease inhibitor
inhibitor IFEDQ 650.29 f107 - kunitz-type
VGVVIQ 613.38 f194 - kunitz-type 111 protease
199 protease inhibitor
inhibitor FEDQL 650.29 f108 - kunitz-type
GTPVRFIGS 932.51 f91 - serine protease 112 protease
99 inhibitor inhibitor
4 LGEMVT 563.26 f25 - patatin LPSDA 501.24 f29 - serine protease
29 33 inhibitor
(<1kDa, GEMVT 535.23 f26 - patatin NSDVGPS 674.29 f84 - serine protease
EGU40) 30 90 inhibitor
GEMVTV 650.29 f26 - patatin SDVGPS 560.24 f85 - serine protease
31 90 inhibitor
IDGGGI 530.27 f34 - patatin EDELL 617.29 f109 - serine protease
39 113 inhibitor
EFLEGQ 721.33 f49 - patatin SDGPEVIE 844.38 f27 - proteinase
54 34 inhibitor I
EVDNNK 717.33 f57 - patatin EVIEL 601.33 f31 - proteinase
62 35 inhibitor I
DVIGGT 560.28 f71 - patatin 17 EVDNNKDARL 1172.58 f57 - patatin
76 66
TEVAIS 618.32 f148 - patatin (1-2 kDa, NLAKSPELDAK 1184.64 f169 - patatin
153 C) 179
AKSPE 530.27 f171 - patatin KSESDYGDVV 1097.49 f67 - Cys protease
175 76 inhibitor
SPELD 559.25 f173 - patatin KSESDYGDVVR 1253.59 f67 - Cys protease
177 77 inhibitor
LVDGA 473.25 f213 - patatin ESDYGDVVR 1038.46 f69 - Cys protease
217 77 inhibitor
VDGAVAT 631.32 f214 - patatin NDEQLVVTGG 1030.49 f103 - Cys protease
220 112 inhibitor
ATVGDPA 629.30 f219 - patatin LQEVDNNKDAR 1300.64 f55 - patatin
225 65
TVGDPA 558.27 f220 - patatin EVDNNKDARL 1172.58 f57 - patatin
225 66
VGDPA 457.22 f221 - patatin EVDNNKDARLAD 1358.64 f57 - patatin
225 68
VGDPAL 570.30 f221 - patatin VDNNKDARLAD 1229.60 f58 - patatin
226 68
GDPAL 471.23 f222 - patatin IGGTSTGGLLTAMITTPNE 1848.91 f73 - patatin
226 91
GTNSE 506.20 f261 - patatin ITTPNENNRPF 1301.64 f86 - patatin
265 96
VQVGE 530.27 f343 - patatin VTHTSNGDKYE 1249.56 f200 - patatin
347 210
LEGQLQE 815.40 f51 - patatin GTGTNSEFDK 1054.46 f259 - patatin
57 268
DYFDVI 770.35 f68 - patatin VSKDSPETY 1024.47 f354 - patatin
73 362
MITTPNE 820.36 f85 - patatin KSESDYGDVV 1097.49 f67 - Cys protease
91 76 inhibitor
NLAKSPE 757.40 f169 - patatin VVTGGKVGNEND 1187.58 f108 - Cys protease
175 119 inhibitor
SPELDAK 758.38 f173 - patatin LVTVDDDKD 1018.48 f163 - Cys protease
179 171 inhibitor
TSNGDKYE 912.38 f203 - patatin 8 VDDDKDFIPF 1209.56 f166 - Cys protease
210 175 inhibitor
SLGTGTNSE 864.38 f257 - patatin (1-2 kDa, VKDNPLDVSFKQ 1388.73 f204 - aspartic
265 EDU40) 215 proteinase
VSKDSPE 760.36 f354 - patatin inhibitor
360 KSPNSDAPCANGIF 1476.67 f68 - aspartic
LPSDA 501.24 f29 - aspartic 81 proteinase
33 proteinase inhibitor
inhibitor VNENPLDVL 1011.52 f208 - kunitz-type
GGDVYLG 679.32 f61 - aspartic 216 protease
67 proteinase inhibitor
inhibitor VKDNPLDVSFKQ 1388.73
(continued on next page)

8
H. Liu et al. Food Chemistry 405 (2023) 134873

Table 3 (continued ) Table 3 (continued )

Fraction Peptide MW Matched sequences* Fraction Peptide MW Matched sequences*
no. (Da) no. (Da)

f208 - serine protease KVNDEQLVVT 1143.61 f101 - Cys protease

219 inhibitor 110 inhibitor
GTPVRFIGSS 1019.54 f91 - serine protease NDEQLVVTGG 1030.49 f103 - Cys protease
100 inhibitor 112 inhibitor
VLFFMILATE 1200.64 f9-18 patatin CKNIGGNFKNGYPR 1623.79 f149 - Cys protease
9 LQEVDNNKDARLA 1484.76 f55 - patatin 162 inhibitor
67 GGNFKNGYPR 1108.54 f153 - Cys protease
(1-2 kDa, MITTPNENNRP 1285.61 f85 - patatin 162 inhibitor
EGU40) 95 NFKNGYPRL 1107.58 f155 - Cys protease
VLQEKLGETR 1171.66 f133 - patatin 163 inhibitor
142 LVTVDDDKD 1018.48 f163 - Cys protease
LQEKLGETRVH 1308.72 f134 - patatin 171 inhibitor
144 VDDDKDFIPF 1209.56 f166 - Cys protease
QEKLGETRV 1058.57 f135 - patatin 175 inhibitor
143 MKCLFLLCL 1212.61 f1 - 9 aspartic
GETRVHQALTE 1239.62 f139 - patatin proteinase
149 inhibitor
RVHQALTEVAIS 1322.73 f142 - patatin AGKELDSRLS 1074.57 f40 - aspartic
153 49 proteinase
DIKTNKPVI 1026.61 f156 - patatin inhibitor
164 DVGPSGTPVRF 1130.57 f86 - aspartic
KSNLAKSPELDAK 1399.77 f167 - patatin 96 proteinase
179 inhibitor
SNLAKSPELDAK 1271.67 f168 - patatin YTIWKVGDYD 1204.47 f124 - aspartic
179 133 proteinase
NLAKSPELDAK 1184.64 f169 - patatin inhibitor
179 LPSDATPVLD 1026.52 f29 - aspartic
AKSPELDAKMY 1267.61 f171 - patatin 38 proteinase
181 inhibitor
AAPTYFPPHY 1162.54 f189 - patatin KDNPLDVSFK 1161.6 f205 - aspartic
198 214 proteinase
APTYFPPHY 1091.51 f190 - patatin inhibitor
198 MKCLFLLCL 1212.61 f2 - 10 kunitz-type
PTYFPPHY 1020.47 f191 - patatin protease
198 inhibitor
TSNGDKYFNE 1173.49 f203 - patatin IISIGRGALGGD 1127.63 f56 - kunitz-type
212 67 protease
FNLVDGAVAT 1005.51 f211 - patatin inhibitor
220 SIGRGALGGDVYLGK 1461.79 f58 - kunitz-type
GTGTNSEFDKTY 1318.57 f259 - patatin 72 protease
270 inhibitor
GTGTNSEFDKTYTAE 1619.70 f259 - patatin IGRGALGGDVYLGK 1374.76 f59 - kunitz-type
273 72 protease
NSEFDKTY 1002.43 f263 - patatin inhibitor
270 KSPNSDAPCPDGVFR 1645.75 f72 - kunitz-type
GEKLLKKPVSKDSPE 1653.93 f346 - patatin 86 protease
360 inhibitor
LKKPVSKDSPETY 1490.80 f350 - patatin DVGPSGTPVRF 1130.57 f90 - kunitz-type
362 100 protease
TYEEALKRF 1155.59 f361 - patatin inhibitor
369 LETGGTIGQADSSYF 1544.70 f145 - kunitz-type
VYDQDGNPL 1019.46 f6 - 14 Cys protease 159 protease
inhibitor inhibitor
YDQDGNPLR 1076.49 f7 - 15 Cys protease MKCLFLLCL 1212.61 f1 - 9 serine protease
inhibitor inhibitor
RKSESDYGDVVR 1409.69 f66 - Cys protease LPSDATPVLDVD 1226.64 f29 - serine protease
77 inhibitor 40 inhibitor
KSESDYGDVV 1097.49 f67 - Cys protease LPSDATPVLDVDGK 1411.76 f29 - serine protease
76 inhibitor 42 inhibitor
KSESDYGDVVR 1253.59 f67 - Cys protease SPNSDAPCANGVFR 1490.66 f69 - serine protease
77 inhibitor 82 inhibitor
KSESDYGDVVRVM 1483.70 f67 - Cys protease SDVGPSGTPVRFIGS 1474.74 f85 - serine protease
79 inhibitor 99 inhibitor
SESDYGDVVR 1125.49 f68 - Cys protease DVGPSGTPVRF 1130.57 f86 - serine protease
77 inhibitor 96 inhibitor
ESDYGDVVR 1038.46 f69 - Cys protease SSHFGQGIFE 1107.50 f100 - serine protease
77 inhibitor 109 inhibitor
ESDYGDVVRV 1137.53 f69 - Cys protease VGDYDASLGTM 1143.48 f133 - serine protease
78 inhibitor 143 inhibitor
ESDYGDVVRVM 1284.57 f69 - Cys protease LPSDATPVLD 1026.52 f29 - serine protease
79 inhibitor 38 inhibitor
SDYGDVVRVM 1139.53 f70 - Cys protease KDNPLDVSFK 1161.60 f209 - serine protease
79 inhibitor 218 inhibitor
DYGDVVRVM 1052.50 f71 - Cys protease
79 inhibitor * From National Center for Biotechnology Information (NCBI).
C: non-ultrasonicated conventional hydrolysis; EDU40: energy divergent

9
H. Liu et al.
ultrasound assisted enzymatic hydrolysis with power density of 40 W/L; EGU40:
energy gather ultrasound assisted enzymatic hydrolysis with power density of
40 W/L.
et al., 2015). There were 5, 3, 13, 10, 7 and 48 peptide sequences with
aromatic amino acid residues phenylalanine (F), tyrosine (Y) or imid­
azole amino acid histidine (H) were identified in peptide fractions from
MW < 1 and 1–2 kDa by C, EDU40 and EGU40 respectively (showed in
bold, Table 3). Furthermore, 1, 1, 4 and 11 peptide sequences in peptide
fractions from MW < 1 and 1–2 kDa by EDU40 and EGU40 contained
sulfur amino acids (M) (bold, Table 3). Only 1 peptide sequence was
detected in MW 1–2 kDa by EGU40 containing tryptophan (W)
(underlined, Table 3). In addition, 1 and 6 peptide sequences with
cysteine (C) were detected from MW 1–2 kDa by EDU40 and EGU40
respectively (underlined, Table 3). Obviously, peptide fractions from
MW < 1 and 1–2 kDa by EGU40 had contained more potential antiox­
idant amino acids of Y, W, F, H, C and M.
3.6.1. Conformation of peptide sequences
According to the amino acids related to antioxidant activity and their
positions, peptide sequences of IFGPM, IDGGGI, HGPHIF,
VDDDKDFIPF, LVTVDDDKD and VVTGGKVGNEND with potential
antioxidant activity were screened (underlined, Table 3), and their
conformations were analyzed (Fig. 3).
The peptide IFGPM had three turns, and contained an aromatic Phe
and 3 hydrophobic amino acids (I, F and M) with I and M located at the
N- and C- terminus (Fig. 3 a). The peptide IDGGGI had three turns with 2
hydrophobic amino acids (I) located in the C- and N-terminal, and
contained one Asp (D) with hydroxyl scavenging ability (Fig. 3 b). The
peptide HGPHIF had two turns, and contained 2 hydrophobic amino
acids (I and F), exposing two imidazole amino acids His (H) and a bulky
hydrophobic aromatic Phe (F) to the external medium (Fig. 3 c). The
Fig. 3. Conformational structures of potential antioxidant peptides from MW 1–2 and <1 kDa fractions of PPH by EDU40 and EGU40. (a) IFGPM; (b) IDGGGI; (c)
HGPHIF; (d) VDDDKDFIPF; (e) LVTVDDDKD; (f) VVTGGKVGNEND.
10
Food Chemistry 405 (2023) 134873
peptide VDDDKDFIPF showed three turns, was closely surrounded by 4
Asp (D), and had 4 hydrophobic amino acids Val (V), Ile (I) and two Phe
(F) exposed to the external medium (Fig. 3 d). The compact peptide
LVTVDDKDD showed four turns, was closely surrounded by 3 Asp (D),
and had one Asp (D) and one Leu (L) located at the C- and N-terminal
(Fig. 3 e). The peptide VVTGGKVGNEND had the most compact struc­
ture with five turns, which contained 3 hydrophobic amino acids (V) and
1 Asp (D) (Fig. 3f). Compactness of peptide structures was an important
indicator of antioxidant capacities of peptides (Habinshuti et al., 2021).
Antioxidant properties of the tightly structured sweet potato peptide
RYYDPL were significantly higher than that of FVIKPT (Zhang & Mu,
2017). The C- and N-terminal hydrophobic amino acids also presented a
strong correlation with antioxidant activity of the peptides (Li & Li,
2013).
4. Conclusion
In this study, EDU and EGU-assisted enzymatic hydrolysis of potato
protein presented a significant impact on enzymatic hydrolysis progress,
antioxidant activities and peptide characteristics as compared to con­
ventional enzymatic hydrolysis. EDU and EGU showed positive effects
on enzymatic hydrolysis progress revealed by DH and recovery of PPH.
EDU and EGU-assisted hydrolysis at a power density of 40 W/L signifi­
cantly increased production of MW < 2 kDa peptides with higher anti­
oxidant activity (82.47 µg TE/mL), and EGU presented better
enhancement effect. Peptide characteristics also confirmed the potential
usefulness of EGU-assisted enzymatic hydrolysis, in terms of stronger
antioxidant activity of MW < 1 and 2 kDa peptides by EGU40, which
was attributed to their higher content of hydrophobic amino acids
located at N- and/or C-terminus, more polar charged amino acids at C-
terminus, and more antioxidant amino acids (Y, W, F, H, C and M) inside
the sequences. Conformational structure revealed that the potential
H. Liu et al.
antioxidant peptides were compact, and potential antioxidant amino
acids exposed to the external. Thus, EGU can be used to improve re­
covery and bioactivity of food protein hydrolysates, making it of great
potential for the production of antioxidant peptides and functional food.
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.
Data availability
Data will be made available on request.
Acknowledgements
The authors gratefully acknowledge the earmarked fund for CARS-
10-Sweetpotato. We also thank the International Cooperation on Po­
tato Processing and Quality Control in “the Belt and Road” Country
(DL2021056002L) and the Science and Technology Innovation Project
of Chinese Academy of Agricultural Sciences (CAAS-ASTIP-202X-IFST).
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