Food Chemistry 270 (2019) 243–250

Contents lists available at ScienceDirect

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

Physico-chemical properties, antioxidant activities and angiotensin-I T

converting enzyme inhibitory of protein hydrolysates from Mung bean
(Vigna radiate)
⁎
Jianhua Xiea,b, Mengxia Dua, Mingyue Shena, , Ting Wua, Lihua Lina,c
a
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China
b
Whistler Center for Carbohydrate Research, Department of Food Science, Purdue University, West Lafayette, IN 47907-2009, USA
c
Environment and Food Engineering School, Liuzhou Vocational & Technical College, Liuzhou 545000, China

A R T I C LE I N FO A B S T R A C T

Keywords: Mung bean Protein hydrolyses (MBPHs) have attracted a great deal of attention due to their variety of biological
Mung bean protein activities. In present study, MBPHs were fractionate according to the molecular mass into three fractions of
Hydrolysates MBPHs-I (< 3 kDa), MBPHs-II (3–10 kDa) and MBPHs-III (> 10 kDa). Their antioxidant activity and angio-
Antioxidant activity tensin-I converting enzyme (ACE) inhibitory of were investigated in vitro. Results showed that the alcalase-
ACE inhibitory activity
derived hydrolysate exhibited the highest degree of hydrolysis (DH) and trichloroacetic acid–nitrogen soluble
Secondary structure
Amino acid
index (TCA-NSI) versus those of other enzyme hydrolysates. MBPHs-I presented the best scavenge DPPH, hy-
droxyl radicals, superoxide radicals, Fe2+ chelating activities, and the best ACE inhibitory activity
(IC50 = 4.66 μg/mL) than that of MBPHs and MBPHs-III. And MBPHs-I rich in hydrophobic and aromatic amino
acids, and its secondary structure mainly contain α-helix, β-sheet and irregular coiled. Results indicated that
MBPHs-I has a great potential as natural functional materials for supplement.

1. Introduction quantities as an alternative to animal proteins in product formulations.

Mung bean (Vigna radiata L.) is popular and important leguminous
crop in Asia and other parts of the world. In China, it has been con-
sumed as a common food for thousands of years. Nowadays, mung bean
is accepted by different people all over the world for its physiological
functionalities, such as antiangiotensin I-converting enzyme (ACE) in-
hibitory, antitumor, antioxidant, and antidiabetic (Randhir & Shetty,
2007; Soucek, Skvor, Pouckova, Matousek, & Slavík, 2005; Yao et al.,
2013). It is rich in vitamins, minerals, proteins, and essential amino
acids, and the mung bean protein is approximately 25–28% on a dry
weight basis (Khaket, Dhanda, Jodha, & Singh, 2015). Much focus has
been placed on researching the properties of mung bean starch rather
than the mung bean protein (MBP) (Ahmed, 2012). The amino acid
profile of mung bean protein shows that it can meets the amino acid
requirements of the human body (Du et al., 2018). In most developing
countries, such as China and India, mung bean protein is used in large
In addition to applied as major protein resource, beans protein can
also produce biological function peptides that have biological proper-
ties, such as antioxidant and anti-inflammatory activity (Connolly,
Keeffe, Piggott, Nongonierma & FitzGerald, 2015). Enzymatic hydro-
lysis can improve functional properties of target protein by translating
it into peptides without affecting its nutritive value (Moure,
Domínguez, & Parajó, 2006). Enzyme treatment has been documented
to be an effective treatment to eliminate antinutritional factors (Kuhad,
Gupta, & Singh, 2011). Commercial enzymes, such as alcalase, pepsine,
trypsin, flavourzyme, protamex, prolyve, and promod usually used for
the hydrolysis of protein.
Dietary peptides with high nutritional value from animal or plant
proteins are reported to have a potential in the regulation of food intake
and body weight in humans (Anderson & Moore, 2004; Hartmann &
Meisel, 2007). Polypeptides may exhibit various biological activities,
such as immunomodulatory, antioxidant, antihypertensive, and

Abbreviations: ACE, antiangiotensin I-converting enzyme; DPPH, 1,1-diphenyl-2-picrylhydrazyl; DH, the degree of hydrolysis; TCA, trichloroacetic acid; FTIR,
Fourier transform infrared; CD, circular dichroism; MBP, Mung bean protein; TCA-NSI, trichloroacetic acid-nitrogen soluble index; HHL, hippuryl-histidyl-leucine;
ALH, alcalase hydrolysates; NTH, neutrase hydrolysate; PAH, papain hydrolysates
⁎
Corresponding author at: State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, Jiangxi,
China.
E-mail addresses: jhxie@ncu.edu.cn (J. Xie), shenmingyue1107@ncu.edu.cn (M. Shen).

https://doi.org/10.1016/j.foodchem.2018.07.103
Received 12 October 2017; Received in revised form 30 April 2018; Accepted 16 July 2018
Available online 17 July 2018
0308-8146/ © 2018 Elsevier Ltd. All rights reserved.
J. Xie et al.
antibacterial effects (Hartmann & Meisel, 2007). Studies over the past
ten years have demonstrated that protein hydrolysates, such as brewers’
spent grain protein (Vieira, da Silva, Carmo, & Ferreira, 2017), peanut
(Jamdar et al., 2010), pea seed (Pownall, Udenigwe, & Aluko, 2010)
have good antioxidant ability. In vitro studies also demonstrated that
protein hydrolysates from other legumes, such as lupin (Boschin,
Scigliuolo, Resta, & Arnoldi, 2014), soy (Chiang, Tsou, Tsai, & Tsai,
2006), Phaseolus lunatus and Phaseolus vulgaris seeds (Torruco-Ucoa,
Chel-Guerrerob, Martínez-Ayalac, Dávila-Ortíza, & Betancur-Anconab,
2009) have good ACE inhibitory and antioxidant ability. However,
studies on the biological activity of hydrolysates with different mole-
cular weight from MBP are rarely reported, and their secondary struc-
ture and the composition of amino acids are still not clear. The aim of
this study is to determine the antioxidant activities and ACE-inhibitory
activity and to analyze the amino acid composition and secondary
structure of MBPHs, MBPHs-I, MBPHs-II, and MBPHs-III obtained from
MBP by ultrafiltration separation.
2. Materials and methods
2.1. Materials
Mung beans (Vigna radiata L.) were purchased from the Grain and
Oil Processing Company (Jilin, China). Briefly, Mung beans were
cleaned, dehulled and ground into flour. Alcalase, neutrase, papain,
protamex were purchased from Novozymes (Denmark). 1,1-diphenyl-2-
picrylhydrazyl (DPPH), ascorbic acid, hippuryl-l-histidyl-l-leucine
(HHL) and ACE (from rabbit lung) were obtained from Sigma Chemical
Company (St. Louis, MO, USA). Other chemicals and solvents used in
this study were of analytical grade.
2.2. Preparation of enzymatic hydrolysates
Protein concentrate hydrolysis from Mung bean was prepared by
individual treatments with alcalase (pH 8.0, 55 °C), neutrase (pH 7.0,
45 °C), papain (pH 7.0, 45 °C), and protamex (pH 5.5–7.55, 60 °C)
(Novo Nordisk, Bagsvaerd, Denmark). The mixture with enzyme/sub-
strate ratio of 3/100 (w/v) was reacted for 2 h then heated in boiling
water bath for 10 min to inactivate the enzyme. After cooling, the
mixture was adjusted to pH 4.2, then centrifuged at 5000×g (4 °C) for
20 min. The supernatant was recycled using decantation and then
loaded into a dialysis bag (Mw, 300 Da) to desalinate for 48 h. Finally,
hydrolysates were lyophilized and stored under −80 °C until next use.
2.3. Selecting the appropriate enzyme
2.3.1. Determination of DH
The extent of proteolytic degradation was by measured by means of
the degree of hydrolysis (DH) according to method described by
Guerard, Dufosse, De La Broise, and Binet (2001). DH values for DH is
defined as the percent ratio of the number of peptide bonds broken (h)
to the total number of peptide bonds in the substrate studied (htot). The
values for DH were calculated as the following equation:
DH (%) = h htot × 100 = (B × Nb) (MP × α × htot ) × 100% (1)
where B = the amount of base consumed (mL) during the reaction.
Nb = the normality of the base, MP = the mass (g) of protein
(N × 6.25), α = the average degree of dissociation of the α-NH2 re-
leased during hydrolysis, and htot is defined as 7.9 meq/g.
2.3.2. Determination of TCA-NSI value
The trichloroacetic acid–nitrogen soluble index (TCA-NSI) was es-
timated by measuring the nitrogen content of hydrolyzed proteins so-
lubilized in 10% trichloroacetic acid (TCA) according to the AOAC
method (Williams, 1984). A total of 10 mL of 10% TCA solution was
mixed with 10 mL of enzymatic hydrolysates, and then the resulting
244
Food Chemistry 270 (2019) 243–250
mixture was centrifuged at 15,000×g for 5 min. The supernatant was
analyzed using the Kjeldahl method (Minagawa, Winter, & Kaplan,
1984) to determine nitrogen content in TCA-soluble peptides and free
amino acid. TCA-NSI was calculated according to the following equa-
tion (Zhang et al., 2012):
TCA−NSI (%) = (N1 N0) × 100 (2)
where N1 is the nitrogen content (in mg) soluble in 20% TCA and N0 is
the total nitrogen content in MBP hydrolysates (in mg).
2.4. Proximate composition analysis
Lyophilized MBP and its hydrolysates were analyzed in terms of
protein content using the Kjeldahl method (Minagawa et al., 1984). The
fat was extracted with chloroform:methanol (2:1, v/v) with a Soxhlet
extractor (Shanghai, China, then determined gravimetrically after oven-
drying (80 °C) overnight. The ash contents of MBP and its hydrolysates
were estimated by heating the overnight in a muffle furnace at 550 °C
(AOAC, 1995), and dry matter content (oven at 105 °C). The contents
were indicated in terms of dry weight (% dw).
2.5. Fractionation of the protein hydrolysates
Freeze-dried MBPHs with a concentration of 100 mg/ml was dis-
solved and further separated using by ultrafiltration cube with 10 and
3 kDa MWCO, resulting in three fractions, namely, MBPH-I (< 3 kDa),
MBPH-II (3–10 kDa) and MBPH-III (> 10 kDa).
2.6. Physicochemical characteristics
2.6.1. UV spectrum
UV–visible spectra of samples were scanned using a spectro-
photometer (TU-1900, Purkinje General Instrument Co., Beijing, China)
in the range of 200–800 nm. Each of the samples was dissolved in
100 mL of distilled water.
2.6.2. Circular dichroism (CD) spectroscopy
CD spectra were obtained on a Bio-Logic MOS 450 CD spectrometer
(Bio-Logic, Claix, France) in the far-UV region (190–250 nm). All CD
spectra were recorded in distilled water at room temperature with a
fixed concentration of MBPHs, MBPHs-I, MBPHs-II, and MBPHs-III; the
distilled water signal was subtracted for baseline correction.
2.6.3. Fourier transform infrared (FTIR) spectroscopy
Proteolytic digestion samples of different molecular segments were
desiccated prior to FTIR analysis. FTIR spectra of the samples were
recorded using Fourier-transform infrared spectrophotometer (Thermo
Electron, USA) from 400 cm−1 to 4000 cm−1 at room temperature. The
measuring resolution was 4 cm−1 and iterations were conducted for 32
times.
2.6.4. Determination of amino acid composition
The amino acid composition of the MBPHs and the three other
fractions was analyzed using a model L-8900 Amino Acid Auto-
Analyzer (L-8900, HITACHI, Japan). First, the sample solution was
filtrated through a 0.22 µm pore size membrane filter and then injected
into the analyzer to determine the free amino acid content. Finally, the
sample was determined at 110 °C for 22 h with 6 M HCl.
2.7. Screening for antioxidant activity of MBP hydrolysates
2.7.1. DPPH radical scavenging activity
The scavenging effects on DPPH free radical of MBP hydrolysates
were determined by Xie et al. (2010, 2015). DPPH solution (1 mL, 1 mM
in 95% ethanol) was mixed with 1 mL of samples. At 25 °C, the mixture
was shaken and left for 30 min, finally the absorbance of the solution
J. Xie et al.
was measured at 517 nm in the microplate reader (Thermo Electron
Corporation, USA). As a positive standard, ascorbic acid was employed.
The scavenging capacity was determined as follows:
Scavenging activity (%) = [1−−A1 A 0 ] × 100% (3)
where A1 = absorbance of sample, A0 = the absorbance of water in-
stead of sample.
2.7.2. Hydroxyl radicals scavenging ability assay
The hydroxyl radicals scavenging activity was determined ac-
cording to the reported procedure of Li, Chen, Wang, Ji, and Wu
(2007). In this procedure, a 1.0 mL sample was mixed with FeSO4
(1.0 mL, 2 mM) and salicylic acid-ethanol solution (1.0 mL, 6 mM).
Afterwards, 1.0 mL of H2O2 (6 mM) was added to the solution before
incubation at 37 °C in water bath for 30 min. Afterward, the absorbance
was instantly tested at 510 nm by adopting a microplate reader; as-
corbic acid was also used as a positive standard. Hydroxyl radical
scavenging activity was calculated using Eq. (4):
Scavenging activity (%) = [1−(A2 −− A1 ) A0 ) × 100% (4)
where A2 = absorbance of sample, A1 = absorbance of sample using
water instead of H2O2, and A0 = absorbance of water instead of sample.
2.7.3. Metal chelating assay
The Fe2+ chelating activities of these fractions were also de-
termined according to following procedures. A total of 100 μL of the
sample fractions in tubes, 135 μL of distilled water, and 5 μL of FeCl2
(2 mM) was added. After 3 min, 10 μL of ferrozine (5 mM) was added.
The mixture was shaken and remained static for 10 min at 25 °C.
Finally, the absorbance of the mixture (A1) was determined at 562 nm
in the microplate reader (Thermo Electron Corporation, USA). The
absorbance of a blank (A0) was measured following the above method
using distilled water. The scavenging capacity was determined by fol-
lows:
Scavenging activity (%) = [1−−A1 A0 ] × 100% (5)
2.7.4. Superoxide radicals scavenging activity
The superoxide radical scavenging activity was determined ac-
cording to the method we reported in our previous study (Xie et al.,
2016). Briefly, 5.0 mL Tris-HCl buffer solution (50 mM, pH 8.2) and
0.5 mL samples solution were mixed at 25 °C in water bath for 20 min.
Afterwards, 0.5 mL pyrogallol (3 mM) incubated at 25 °C was added
quickly and the absorbance of the mixture was punctually tested every
30 sat a time at 325 nm in 5 min. The change speed of absorbance (A/
min) of the reactive solution was measured at 325 nm every 10 s for
5 min, against a blank (distilled water). The scavenging activity was
counted adopting the formula.
Superoxide anion scavenging activity (%) = (1−−A1 A0 ) × 100% (6)
where A0 is the change speed of absorbance of the blank group (distilled
water) in the superoxide radical generation system and A1 is the change
speed of absorbance of the sample.
2.7.5. Reducing power
The reducing power was analyzed using the method described by
Oyaizu (1986), with a minor modification. A total of 100 μL of samples
were separated in the tube, then phosphate buffer (100 μL, 0.2 M, pH
6.6) and potassium ferricyanide (100 μL, 1%) was added. Next, 100 μL
of 10% TCA was added into this reaction mixture and incubated at
50 °C. An aliquot of 144 μL from the mixture and 144 μL of distilled
water with of 25 μL of 0.1% FeCl3 were mixed in an eppendorf tube.
The absorbance of the solution was determined at 700 nm. Ascorbic
acid was also used as a reference. Absorbance (A700) of the reaction
mixture represented reducibility.
245
Food Chemistry 270 (2019) 243–250
2.8. Measurement of ACE inhibitory activity
The ACE inhibition activity was assayed according to the method
reported by Nakamura, Suzuki, and Hiromi (1995). A total of 40 μL
protein hydrolysates were added into a 5 mL tube containing HHL, then
it was preincubated (3 min, 37 °C). Then, the reaction was started by
putting ACE (20 μL, 0.1 U/mL) in the tube and was incubated (30 min,
37 °C). A total of 250 μL of 1 mol/L HCl was added to stop the enzyme
reaction. Ethyl acetate was added then mixed with strong shock; a total
of 1 mL of ethyl acetate was separated and baked at 90 °C in the oven.
After 1 h of drying, the final addition of 4 mL distilled water was fully
dissolved and the absorbance value was detected at the same time. In
addition to 250 μL of HCl added in the control tube, the rest of the steps
were the same; the blank tube and captopril were used as reference
because of their good ACE activity.
ACE Inhibition rate (%) = (Ab −−Aa ) (Ab − Ac ) × 100% (7)
Aa is the absorbance value of samples and ACE enzyme reaction; Ab
is the absorbance value of the water instead of the sample and ACE
enzyme reaction without sample solution; Ac is the absorbance value of
the bank group. Before the reaction, the ACE was inactivated and then
added into the sample solution, which was the blank control value of
ACE and HHL reaction.
2.9. Statistical analysis
All experiments were conducted three times and the data were
means of three values. Origin Pro (version 8.0) software (Stat-Ease, Inc.,
Minneapolis, USA) were utilized for analyzing results. Analysis of var-
iance was carried out by ANOVA Duncans new multiple range test was
carried out to define the significance differences using SPSS 13.0 soft-
ware (SPSS Inc., Chicago, USA). The P ≤ 0.05 was defined as significant
differences between the samples.
3. Results and discussion
3.1. Preparation of the MBP hydrolysates
DH is a very important value that can influence the molecular sizes
and amino acid compositions of the peptides, hence affecting the bio-
logical activities of the peptides (Jamdar et al., 2010). TCA-NSI can
evaluate the low molecular weight amino acids and free amino acids
content, and infer the degree of DH. In this study, the DH and TCA-NSI
value were used as reference indicators for the hydrolysis capacity of
the MBP. In the current study, alcalase, neutrase, papain, and protamex
were used to obtain hydrolysates from MBP. The DH and TCA-NSI va-
lues of the resulting hydrolysates are shown in Fig. 1.
The hydrolysis curves of the MBP are shown in Fig. 1(a). Four dif-
ferent proteolytic enzymes, including alcalase, neutrase, papain, and
protamex were used to compare the hydrolytic efficiency. The hydro-
lysis of proteins was featured at a high rate for the first 1 h; the enzy-
matic reaction was stable. After 2 h of hydrolysis, the alcalase hydro-
lysate attained the highest DH value of 23.51% ± 0.57%, followed by
the neutrase hydrolysate (20.77% ± 0.21%), however the papain hy-
drolysate reached a lower DH value of 15.04% ± 0.63%. The trend of
DH was same to the previous reports for the hydrolysates from P. lu-
natus and P. vulgaris seeds (Torruco-Uco et al., 2009) and soy (Chiang
et al., 2006), chickpea protein (Pedroche et al., 2002).
The TCA-NSI value of the MBPHs and the other three fractions are
shown in Fig. 1(b). Alcalase hydrolysates (ALH) exhibited significantly
higher TCA-NSI value (38.34% ± 0.65%), followed by the neutrase
hydrolysate (NTH) (33.23% ± 0.45%), whereas the lowest TCA-NSI
value were observed in the papain hydrolysates (PAH) all the time. The
higher TCA-NSI indicated higher content of oligopeptide (Tian et al.,
2015). This data trend was consistent with the DH curves of the four
hydrolysates. ALH can be used for follow up studies because of the
J. Xie et al.
Fig. 1. (a) Degree of hydrolysis (DH), and (b) Trichloroacetic acid-nitrogen
soluble index (TCA-NSI) of MBPHs obtained with Alcalase, Neutrase, Protamex,
Papain.
results showed above.
3.2. Proximate composition of the MBP and its hydrolysates
The proximate composition of the MBP and its hydrolysates
(MBPHs) obtained by the alcalase treatment is presented in Table 1S
(Supplementary material). The protein content of MBPHs was slightly
lower than that of the MBP, which may be attributed to the increase in
free amino acid. MBPHs presented the highest content of ash
(6.62% ± 2.89%), presumably caused by the buffer required for the
preparation of the MBP extract rich in proteases. MBPHs also presented
significantly lower fat content (0.10%) than the hydrolysates Elsa F.
Vieira has reported in which the fat content of BSYH, NTH, ALH were
2.42%, 3.84%, and 4.59%, respectively (Vieira et al., 2017). High
protein content and low fat content of the MBPHs are contributes to
their use in commercial preparations, namely, for supplementation of
sports nutrition products, weight-loss diets, and other clinical applica-
tions, such as treatment of ulcerative colitis and Crohn’s disease.
3.3. Physicochemical characteristics
3.3.1. UV spectrum
Fig. 2(a) showed ultraviolet spectra of the alcalase hydrolysates
(MBPHs) and the ultrafiltrated permeates, MBPHs-I (< 3 kDa), MBPHs-
II (3–10 kDa) and MBPHs-III (> 10 kDa) in the range of 200–800 nm.
246
Food Chemistry 270 (2019) 243–250
Fig. 2. (a) Ultraviolet spectra and (b) Circular dichroism (CD) spectra of the
protein hydrolysates (MBPHs, MBPHs-I, MBPHs–II and MBPHs–III).
The spectra of these hydrolysates were quite similar, with a weak ab-
sorption at 270 nm. This result indicated that the solution still con-
tained protein.
3.3.2. CD spectra
As shown in Fig. 2(b), one negative band at around 198 nm and
positive bands with small and wide at around 220 nm were observed,
which were the characteristic absorption peak of irregular coiled con-
formation. A-helical peptides with minimum at 222 nm and 208 nm,
and a maximum at 195 nm, which resulted from the maximum at
195 nm, transfer for the α-helix peptide bonds (Wang, Zhang, Yan, &
Gong, 2014). Despite the different molecular weights of the four com-
ponents, huge change in the CD intensities at bands was not observed.
3.3.3. FTIR spectroscopy
FTIR is a kind of molecular absorption spectrum, which can reflect
the energy level transition of molecular vibrations (Abbott, Wolf, Wu,
Butterfield, & Kleiman, 1991).
FTIR spectra of enzymatic hydrolysates of the different molecular
segments were similar (Fig. 3), which indicated that the structure of the
MBPHs and the other three fractions did not change. In addition to
presence of amide groups in the characteristic absorption peak, the
structure of the enzymatic hydrolysates may also contain C]C, C]C,
C]N, C]N, eCOOH, and eOH. In the use of infrared spectrum ana-
lysis of the secondary protein structure, it can be manifested through its
amide I and III belt to determine the peak of its spatial structure
(Carbonaro & Nucara, 2010). The hydrogen bond properties between
C]O and NeH in the amide I belt (1600–1700 cm−1) determined the
vibrational frequency; the wavelength range of 1650 cm−1 to
1658 cm−1 in the amide I belt represented the α-helix characteristic
absorption peak, 1610–1640 cm−1 for the β-sheet structure,
1640–1650 cm−1 for the random coil characteristic absorption peak,
and 1660–1695 cm−1 for the β-turn of characteristic absorption peak.
J. Xie et al. Food Chemistry 270 (2019) 243–250

followed by MBPHS-III (37.72%). MBPHs and its ultrafiltration com-

Fig. 3. Fourier transform infrared (FTIR) spectra of the protein hydrolysates
(MBPHs, MBPHs-I, MBPHs–II and MBPHs–III).
ponents were richer in amino acid than the hydrolysates of chickpea
(Cicer arietinum) (Borrás-Almenar, Clemente-Juan, Coronado, &
Tsukerblat, 1999) and wheat (Tang, Sanville, & Henkelman, 2009).
MBPHs and its ultrafiltration products had a balanced amino acid
composition making it is the most suitable protein resource in human
nutrition (FAO, 1991).
Increase of hydrophobic amino acids may improve the lipid solu-
bility of peptides, which can enhance the antioxidant activity (Sarmadi
& Ismail, 2010). Residues such as Asp, Ser, Gly, Thr, and Val in the
sequences of different peptide fractions obtained by alcalase and fla-
vourzyme, possessed good ACE-I inhibitory activity (Megías et al.,
2009). These amino acids may exist in the peptides and may generate
the ACE-I inhibitory activity. MBPHS-I also contained more aromatic
amino acids (10.57%) than aromatic groups, such as histidine, tyrosine,
and phenylalanine, converting free radicals to stable molecules by
electron donating action, which may lead to a higher biological activity.

3.4. Determination of the antioxidant activity

The wavelength ranges from 1290 cm−1 to 1330 cm−1 in amide III
(1200–1330 cm−1) represented α-helix characteristic absorption peak,
3.4.1. DPPH scavenging activity
1220–1250 cm−1 for the β-sheet absorption peaks, 1265–1295 cm−1
The scavenging activities of all the samples were concentration
for the β-turn absorption peak, and 1245–1270 cm−1 for the random
dependency and showed higher scavenging ability with increase in
coil. In addition, the absorption peak of the amide II band was at
concentration, as shown in Fig. 4. The DPPH scavenging activity of the
1600–1500 cm−1, belonging to the deformation of NeH.
MPBHs-I (74.23%) was close to the synthetic antioxidant ascorbic acid
According to the information provided in Fig. 3, amide I pre-
(85.62%) at 2.6 mg/mL concentration. At lower concentrations, the
dominantly, arose from α-helix (1548.33 cm−1) and β-sheet
MPBHs and MPBHs-III showed higher DPPH activity than MBPHs-II.
(1638 cm−1, 1687 cm−1), while the NeH bending vibrations arose at
Low molecular fractions from cod (Gadus morhua) protein hydrolysates
1548.33 cm−1, which can be attributed to the amide II. A complex mix
reported by Yang, Ho, Chu, & Chow (2008) also exhibited good
of α-helix was obtained at 1315.31 cm−1 and β-sheet (1241.86 cm−1).
scavenging ability. Enzymatic protein hydrolysate with a molecular
Thus, MBPHs and other fractions still contained α-helix and β-sheet in
weight of 3 kDa from Pea Seed (Pisum sativum L.) possessed stronger
the secondary structure. The β-turn structures have been digested and
DPPH scavenging activity (Pownall et al., 2010).
opened by enzymolysis.

3.4.2. Hydroxyl radicals scavenging ability assay

3.3.4. Determination of amino acid composition The hydroxyl radical is the most reactive free radical in biological
The amino acid composition of the MBPHs and its separated com- tissues proteins (Asghar, Raman, & Daud, 2015) and is an active in-
ponents were different, but they were rich in aspartic acid (Asp), glu- itiator for peroxidation of lipids (Srinivasan, 2014). The hydroxyl ra-
tamic acid (Glu) and proline (Pro), as shown in Table 1. The contents of dicals generating system was attributed to the participation of the
hydrophobic amino acids (38.32%), such as glycine (Gly), tyrosine Fenton’s reaction. Hydroxyl radical scavenging ability of all the tested
(Tyr), valine (Val), methionine (Met), phenylalanine (Phe), isoleucine samples showed a concentration-dependent manner. The hydroxyl ra-
(Ile), leucine (Leu) and proline (Pro), in the MBPHS-I were high, dical scavenging effects of the MPBHs-I showed a slightly lower value
than that of ascorbic acid, as shown in Fig. 4(b).
Table 1
Amino acid composition of MPBHs, MBPH-I, MBPHs-II and MBPHs-III. 3.4.3. Fe2+-chelating activity
Coding Amino acid g/100 g
Fe2+ can accelerates the lipid peroxidation chain reaction by gen-
erating OH− by the Fenton reaction (Stohs & Bagchi, 1995). Thus, the
MBPHs MBPHs-I MBPHs-II MBPHs-III chelation of metal ions is especially important, which can greatly re-
duce lipid levels in vivo. Dose-dependent Fe2+-chelating activity was
1 Aspartic (Asp) 9.580 11.292 9.525 10.021
2 Threonine (Thr) 3.022 3.319 2.287 3.236
also observed, as shown in (Fig. 5a). MPBHs-I had the best scavenging
3 Serine (Ser) 5.781 6.618 4.411 5.980 free radical ability (70%) among all components, lower than that of
4 Glxa 20.251 22.231 18.275 18.876 ascorbic acid (93.12%). MPBHs-I displayed the highest Fe2+-chelating
5 Alanine (Ala) 3.964 4.615 3.133 3.943 activity, which was below 1.5 mg/mL. No significant difference
6 Cysteine (Cys) 10.307 10.059 10.029 10.251
(p > 0.05) of Fe2+-chelating activity was observed between the
7 Valine (Val) 4.807 5.499 5.438 4.781
8 Methionine (Met) 1.107 1.650 1.242 1.101 MPBHs-I and MPBHs-II at 3.0 mg/mL I. However, MPBHs showed the
9 Isoleucine (Ile) 3.816 4.928 4.447 3.795 lowest Fe2+ chelating value regardless of the high or low concentration.
10 Leucine (Leu) 7.317 9.151 7.940 7.278
11 Tyrosine (Tyr) 3.072 4.653 3.511 3.056 3.4.4. O2%−-chelating activity
12 Phenylalanine (Phe) 5.831 6.913 6.230 5.799
13 Histidine (His) 3.287 3.044 4.447 3.269
The difference in O2%− scavenging activity was not significant
14 Lysine (Lys) 6.558 6.913 7.616 6.522 (p > 0.05) in the MBPHs, MBPHs-I, MBPHs-II and MBPHs-III at a
15 Arginine (Arg) 7.251 7.325 9.183 7.212 concentration of 1.0 mg/mL. However, at higher concentrations
16 Proline (Pro) 4.047 3.790 2.287 4.879 MBPHs-I showed comparatively higher O2%− scavenging activity in
17 HAAb 32.904 38.317 34.030 37.723
than the MBPHs-II. MBPHs and MBPHs-III displayed slightly lower
18 AAAc 8.903 10.566 9.741 8.855
O2%− scavenging activity than MBPHs-I. MBPHs and MBPHs-III pos-
a
Two amino acids called Glutamic (Glu) and Glutamine (Gln). sibly contained several inactive peptides, thus resulted in reduced
b
Hydrophobic amino acids. whole O2%− scavenging activity.
c
Aromatic amino acids. The fractionated peptides had stronger superoxide scavenging

247
J. Xie et al.
Fig. 4. In vitro antioxidant activities of the MBPHs, MBPHs-I, MBPHs-II and
MBPHs-III. (a) DPPH radical scavenging activity, (b) Hydroxyl radicals
scavenging ability assay. Results are the mean values of triplicate determina-
tions on the sample ± standard deviations.
activity the MBPHs. MBPHs-I had the highest superoxide scavenging
activity, which was likely to be attributed to the higher concentrations
of aromatic amino acids (Table 1). The superoxide scavenging activity
of the fractions was closely correlated with the content of hydrophobic
amino acid, in agreement with a previous result (Li, Jiang, Zhang, Mu,
& Liu, 2008). Studies demonstrated that strong O2%− scavenging ac-
tivity of an alfalfa leaf hydrolysate was caused by their richer special
amino acid composition than alfalfa leaf protein (Xie, Huang, Xu, & Jin,
2008). The unique composition and sequence of amino acid residues
may play an important role in the antioxidant activity.
3.4.5. Reducing power
The magnitude of the reducing ability of the tested samples can
show its potential antioxidant (Wang, Gao, Zhou, Cai, & Yao, 2008).
Compared with MBPHs and other fractions, MBPHs-I showed sig-
nificantly higher reducing power (p < 0.05), but lower than that of
ascorbic acid at all concentrations (Fig. 5c). No significant difference
(p > 0.05) in the scavenging activity between MBPHs-II and MBPHs-III
was observed, but both were higher than that of MBPHs.
3.5. ACE inhibitory activity
ACE activity can lead to an elevated blood pressure in humans, thus
248
Food Chemistry 270 (2019) 243–250
ACE inhibitors are often used as drugs to treat hypertension. Peptides
exhibiting ACE inhibitory activity that are obtained from food proteins
are deemed to be safer than peptides drugs (Hartmann & Meisel, 2007).
Peptides have more advantages, such as their multifunctionality and
easy absorption in the gastrointestinal tract. Therefore, further studies
are needed to elaborate the ACE inhibitory activity of the MBPHs and
other fraction. The inhibitory activity of these preparations is presented
in Fig. 5(d).
The ACE inhibition ratio by MBPHs-I was around 71.22% at the
concentration of 25 μg/mL, which was close to the inhibitory activity of
captopril at concentration of 2.5 ng/mL and other fraction at various
concentrations of 54.12–62.78%. Further digestion increasing to a
concentration of 25 μg/mL did not lead to an increase in the ACE in-
hibition. The results showed that the inhibition capacity increased with
the increase in the concentration at a certain concentration range, as
similar to the reported by other researchers (Lee, Kim, Park, Choi, &
Lee, 2004; Sheih, Fang, & Wu, 2009). The MBPHs-I had the best ACE
inhibitory activity (IC50 = 4.66 μg/mL) than that of MBPHs
(IC50 = 20.37 μg/mL), MBPHs-II (IC50 = 10.18 μg/mL) and MBPHs-III
(10.27 μg/mL). The IC50 value of MBP hydrolysates was lower than
those of hydrolysates from β-conglycinin and glycinin hydrolysates,
which presented IC50 greater than 120 μg/mL (Kuba, Tana, Tawata, &
Yasuda, 2005). Results showed that the protein hydrolysates from
mung bean, especially MBPHs-I components, have high ACE activity,
which is consistent with the results reported by Byun and Kim (2001)
and Fujita, Yamagami, and Ohshima (2001).
Peptides with exhibiting ACE-I inhibitory activity, may be resistant
to digestion by gastrointestinal proteases and absorbed in the small
intestine (Matsufuji et al., 1994). MBPHs-I that exhibited better ACE-I
inhibitory activity may have the ability to resist gastrointestinal pro-
teases and may be suitable for application in food systems that are
meant to benefit people with high blood pressure.
4. Conclusion
This study investigated the physico-chemical properties, antioxidant
and ACE inhibitory activities of MBPHs obtained by treatment with
various enzymes. MBPHs were fractionated into MBPHs-I, MBPHs-II,
and MBPHs-III using an ultrafiltration membrane system. Results
showed that MBPHs obtained by treatment with alcalase presented a
higher DH and TCA-NSI than that obtained by neutrase, papain, and
protamex. MBPHs and their corresponding fractions, particularly
MBPH-I possessed strong DPPH radical scavenging activity, hydroxyl
radical scavenging ability, and metal-chelating activity. MBPHs-I
showed higher ACE-I inhibitory and antioxidant activities in most of the
in vitro assays, which may be caused by the relatively high con-
centrations of the hydrophobic amino acids in the composition of aro-
matic amino acids. The hydrolysates, especially the MBPHs-I from MBP,
may be good dietary supplement to prevent oxidative related diseases.
Acknowledgments
The authors gratefully acknowledge the financial supports by the
Natural Science Foundation of Jiangxi Province, China (No.
20172ACB21004), the Natural Science Fund for Distinguished Young
Scholars of Jiangxi Province, China (No. 20172ACB2134), the program
of the Science Funds of Educational Commission of Jiangxi Province,
China (No. GJJ14144), the Key Research Foundation of State Key
Laboratory of Food Science and Technology, Nanchang University,
China (No. SKLF-QN-201501), and the Innovation Fund of Nanchang
University, China (No. CX2016228).
Conflicts of Interest
The authors declare no conflict of interest.
J. Xie et al.
Fig. 5. In vitro antioxidant and ACE-inhibitory activities of the MBPHs, MBPHs-I, MBPHs-II and MBPHs-III. (a) Iron chelating activity, (b) Superoxide chelating
activity, (c) Reducing power, and (d) ACE-inhibitory activity. Results are the mean values of triplicate determinations on the sample ± standard deviations.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.foodchem.2018.07.103.
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