applied
sciences
Article
Isolation, Purification and Characterization of Proteins in
“Señorita” Banana (Musa acuminata (AAA) ‘Señorita’) Pulp
with Bioactive Peptides Exhibiting Antihypertensive and
Antioxidant Activities
John Matthew Ferreras 1, *, Mia Claire Marie Clemencia 1 , Amelia Hizon-Fradejas 1 , Lawrence Yves Uy 1,2
Mary Ann Torio 1






Citation: Ferreras, J.M.; Clemencia,
M.C.M.; Hizon-Fradejas, A.; Uy, L.Y.;
Torio, M.A. Isolation, Purification and
Characterization of Proteins in
“Señorita” Banana
(Musa acuminata (AAA) ‘Señorita’)
Pulp with Bioactive Peptides
Exhibiting Antihypertensive and
Antioxidant Activities. Appl. Sci.
2021, 11, 2190. https://doi.org/
10.3390/app11052190
Received: 21 September 2020
Accepted: 29 October 2020
Published: 3 March 2021
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Copyright: © 2021 by the authors.
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Attribution (CC BY) license (https://
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4.0/).
Appl. Sci. 2021, 11, 2190. https://doi.org/10.3390/app11052190
and
1 Institute of Chemistry, College of Arts and Sciences, University of the Philippines-Los Baños,
Laguna 4031, Philippines; mlbercansil@up.edu.ph (M.C.M.C.); abhizonfradejas@up.edu.ph (A.H.-F.);
lcuy1@up.edu.ph (L.Y.U.); motorio@up.edu.ph (M.A.T.)
2 Department of Science and Technology-Science Education Institute, 2nd Level, Science Heritage Building,
Sibol St. DOST Compound, General Santos Avenue, Bicutan, Taguig City 1631, Philippines
* Correspondence: jcferreras@up.edu.ph
Abstract: Banana is one of the most important crops, providing multiple benefits. Although it
has been widely studied for its health benefits, little information can be found about its proteins.
This study determined the antihypertensive and antioxidant activities of the crude, purified, and hy-
drolyzed protein extracts from ‘Señorita’ banana pulp. Crude proteins were extracted using Tris-HCl
buffer and purified through ammonium sulfate precipitation, dialysis, and gel filtration chromatog-
raphy. The protein content of the crude, partially purified, and purified extracts were found to be
167.32, 120.45, and 28.51 µg·mL−1 , respectively, with major protein having an approximate molecular
weight of 15 kDa. These extracts were then subjected to enzymatic hydrolysis for release of bioactive
peptides prior to ACE inhibitory and antioxidant activities determination. Among these samples,
the undigested crude extract had the highest ACE inhibitory activity (85.20%). There was also an
observable increase in ACE inhibition of the digested samples with increased digestion time. Mean-
while, the 3-h and 4-h crude digests had the highest DPPH radical scavenging activity with 30.82%
and 34.74%, respectively. These were not significantly different from the activity of the standard,
ascorbic acid. A general decrease in DPPH radical scavenging activity of the samples was observed
with increased digestion time. These observations were coherent with the in silico analysis of the
putative major protein, lectin, which showed that its enzymatic hydrolysis releases ACE inhibitor
and antioxidant peptides.
Keywords: antihypertensive; antioxidant; banana proteins; bioactive peptides; protein extrac-
tion protocol
1. Introduction
Banana is an important food crop worldwide, together with rice, wheat, and corn [1].
It contains high amounts of vitamin B6 , carbohydrates and potassium along with moderate
amounts of vitamin C, manganese, and dietary fiber [2]. Given the high consumption of the
fruit worldwide, various studies associated to banana consumption have been initiated to
prove its health benefits, such as its potent antioxidant, anti-diabetic, hypocholesterolemic
and antihypertensive activities. Most of these are found to be associated in its phenolic
compounds, carotenoids, flavonoids, biogenic amines, and phytosterols while only few
being protein related [3].
Protein studies require samples with high protein content [4]. Despite the high avail-
ability of bananas [1], its low protein content (approx. 1% of the fruit pulp) and the presence
of interfering compounds in higher amounts (e.g., 20% carbohydrates) makes it a difficult
https://www.mdpi.com/journal/applsci
Appl. Sci. 2021, 11, 2190 2 of 13

subject for protein studies, thereby, limiting the nature and number of available studies
that can be cited up to date [5]. Moreover, most of these studies focus on optimization
and comparison of different protein extraction procedures rather than the identification of
isolated proteins.
Extracted crude proteins can be subjected to purification, characterization, and other
protein studies in which bioactive peptide researches are included. Bioactive peptides are
contained in their precursor proteins and can be released through proteolytic processes.
Once released, they can act as potential metabolic regulatory compounds with hormone-
like activity. Activities related to bioactive peptides include antimicrobial, blood pressure
lowering, antioxidant, and atherosclerotic activities [6–8].
Antihypertensive activity is one of the most studied activities associated with bioactive
peptides. According to World Health Organization [9], hypertension causes 7 M deaths
yearly and affects 1.5 billion people around the world that suffering from its complications.
In the Philippines, 21% of Filipino adults are hypertensive which is expected to increase
by the coming years [10]. Studies on selected commercially available antihypertensive
drugs like enalapril showed that they have negative side effects such as kidney damage,
cough, diarrhea, and skin rashes [11]. Angiotensin-converting enzyme (EC 3.4.15.1) (ACE)
catalyzes conversion of angiotensin I to angiotensin II, a vasoconstrictor, making it a target
of inhibition of antihypertensive compounds. Since commercially available drugs exhibit
negative side effects, there are now studies pursuing antihypertensive compounds obtained
from natural resources that will not exhibit negative side effects.
Another health benefit associated with bioactive peptides is their antioxidant activ-
ity. Oxidative stress refers to an imbalance between the free radical production and the
body’s ability to combat its harmful effects [12]. Free radicals and reactive oxygen species
(ROS) generated by different metabolic processes and environmental stress can damage
biomolecules and modify their functions leading to cellular dysfunction and cell death.
These are manifested in the form of health problems such as cancer and accelerated cell ag-
ing [13]. Human tissues are protected against these reactive compounds by endogenous and
exogenous antioxidants from a natural or synthetic origin [14]. One mechanism in which
these delay or inhibit oxidation processes is through interfering with the nonbeneficial
chain reactions caused by free radicals and ROS. Studies on natural antioxidants showed
that their activity are comparable to the activity of the synthetic ones with the advantage of
being cheap and readily available in the diet [15]. Natural antioxidants were also being
studied for their prophylactic and therapeutic properties which can serve as a possible
countermeasure for radiation, combating cancer and other age-related diseases [16].
Given that there are only few studies which can be cited on banana proteomics and
bioactive peptides, this study aims to provide preliminary data on ‘Señorita’ banana protein
extraction, purification, and characterization methods, which may serve as a foundation
for further banana protein studies. Second, it aims to determine the antihypertensive and
antioxidant activities of its protein extracts to prove health beneficial effects associated to
banana consumption.

2. Materials and Methods

2.1. Sample Preparation and Protein Extraction
The ‘Señorita’ banana sample obtained from the local market was peeled and the
pulp was chopped into tiny pieces, flash frozen in liquid nitrogen, and powdered using
mortar and pestle. The crude proteins from the powdered pulp were extracted according
to procedure by Esteve et al. [17] with few modifications. Forty grams of the powdered
pulp was mixed with 80.00 mL of cold extraction buffer (0.125 M tris-HCl [pH 7.4] with
50 mM NaCl) and was stirred overnight, with the container submerged in an ice bath.
The resulting mixture was centrifuged in a refrigerated centrifuge (Z 326K centrifuge,
Hermle, Wehingen, Germany) for 30 min at 4 ◦ C, 56,448× g. Upon separation of the layers,
the supernatant (fraction containing the crude protein extract) was collected and stored in
the freezer with the temperature at approximately −20 ◦ C while the residue was discarded.
Appl. Sci. 2021, 11, 2190 3 of 13

2.2. Purification of the Protein Isolate

2.2.1. Ammonium Sulfate Precipitation/Fractionation
The percent ammonium sulfate saturation of the solution in which most of the proteins
will precipitate was determined first. Percent saturations used were 0–20%, 20–40%,
40–60%, 60–80% and 80–95%. Then, the protein extract was saturated up to the optimized
percent saturation, 40% to 60%, was gently stirred for 30 min in an ice bath, and was
centrifuged at 10,000× g rpm for 30 min at 4 ◦ C. After centrifugation, the precipitate was
collected while the supernatant was discarded.

2.2.2. Dialysis
The precipitate obtained from ammonium sulfate precipitation was dissolved in
minimum amount of extraction buffer and placed in a dialyzing bag with a molecular
weight cut-off of 10 kDa. The dialysis was done for 12 h against distilled water with stirring
in a beaker submerged in an ice bath. The distilled water used was changed every 3 h.

2.2.3. Gel-Filtration Chromatography

A column containing Sephacryl® S-100 as resin was pre-equilibrated and eluted with
extraction prior to loading of the dialyzed samples. The samples were eluted at a flow rate
of 2.00 mL/min. The resulting sixty 1.5 mL fractions were collected and the absorbance at
280 nm of each fractions were determined.

2.3. Protein Quantification Using Colorimetric Bradford Assay

The protein content of the protein extracts was calculated from a standard protein
curve of bovine serum albumin (BSA) using a colorimetric Bradford assay [18]. Five µL
of sample protein extract was added to 250 µL of 1× Bradford reagent in every well of a
300 µL capacity microtiter plate. The resulting solutions were incubated for 5 min with
interval shaking in a UV-Vis microtiter plate holder, before the absorbance was determined
at 595 nm.

2.4. Enzymatic Protein Digestion

The crude, partially purified and purified protein extracts were hydrolyzed while
being submerged in a water bath with shaker at 37 ◦ C using two sets of freshly prepared
enzymes: Set A consists of pepsin while set B consists of trypsin, chymotrypsin and
thermolysin. For the first part of protein digestion, pepsin was added to the sample and
the digestion was allowed to proceed for two hours at pH 2.0. Then, Set B of enzymes
were added, and the digestion was allowed to proceed for additional 1, 2, 10 and 22 h at
pH 7.0. At every specified time, a 2.0 mL aliquot was obtained from the extracts followed
by boiling for 5 min in a boiling water bath to terminate the digestion process. After boiling,
the aliquot was stored to freezer prior to activity testing.

2.5. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS-PAGE was performed using the method developed by Laemmli [19]. The run was
carried out in a 15% discontinuous denaturing stacking and resolving gels using BIORAD
tetracell electrophoresis apparatus. The electrophoretic run was completed for 60 min at 110
V. The gel was stained using staining solution 0.10% (w/v) Coomassie Brilliant Blue R-250,
50% (v/v) methanol, 10% (v/v) acetic acid) for 1 h with shaking and was destained using
destaining solution (50% (v/v) methanol, 10% (v/v) acetic acid). The molecular weights of
the subunits were estimated using a Benchmark Protein Ladder with a molecular weight
(MW) range of 10–250 kDa.

2.6. Densitometric Analysis

Molecular weights of the proteins present in the crude, partially purified, purified
extracts, and enzyme digests were estimated from the SDS-PAGE profiles using the TotalLab
software. The same software was used to determine the extent of protein hydrolysis in
Appl. Sci. 2021, 11, 2190 4 of 13

the digested protein extracts. Peak areas and band volume of the protein bands were
also determined.

2.7. In Silico Analysis of the ‘Señorita’ Banana Major Protein

The amino acid sequence of the putative major protein lectin (E9NX13) extracted from
‘Señorita’ banana was obtained from http://www.uniprot.org/ (retrieval date: February
2019) [20]. The peptides released when the major protein was subjected to simulated
digestion with Pepsin (EC 3.4.23.1), chymotrypsin (EC 3.4.21.1), Trypsin (EC 3.4.21.4)
and thermolysin (EC 3.4.24.27) was determined using the protein sequence analyzer in
http://www.uwm.edu.pl/biochemia/index.php/en/biopep (retrieval date: February
2019) [21]. The same site was used for the determination of the bioactive peptides exhibiting
antioxidant and antihypertensive activities.

2.8. Determination of the Percent (%) Angiotensin Converting Enzyme (ACE) Inhibition
The ACE activity is determined using the method described by Cushman et al. [22]
with few modifications. Initially, 100 µL of HHL buffer (5 mM hippuryl-L-histidine-
L-leucine [HHL] in 0.1 M phosphate buffer [pH 8.2] with 0.3 M NaCl) was placed in
different 2.0 mL Eppendorf tubes. For the sample replicates (undigested and digested
crude, partially purified, and purified protein extracts), 25 µL of the sample was added
to the HHL buffer in the Eppendorf tubes. For the method blank, 25 µL of enzyme blank
(mixture containing only the enzymes used for digestion) was added. For the positive
control, 25 µL of 50 µg/mL captopril solution was added. For the main control (full reaction
control where ACE was not inhibited), only 25 µL of the extraction buffer was added. The
resulting mixtures were incubated in a water bath at 37 ◦ C for 4 min with occasional
stirring in an incubation chamber. Then, 25 µL of purified ACE was added to the sample
tubes except for the method blank. For the method blank, 125 µL of 1.0 M HCl was added.
The resulting mixtures were incubated again in a water bath at 37 ◦ C for 30 min. After
incubation, 125 µL of 1.0 M HCl was added to the mixtures (sample replicates, positive
control and main control) to terminate the reaction, while for the method blank, 25 µL of
ACE was added. Afterwards, 750 µL of ethyl acetate was added to all the sample tubes and
the mixture was stirred using vortex mixer. The resulting mixtures were allowed to stand
under room temperature condition until there was a visible separation of layers. The top
layer of the mixture was obtained and placed in another set of empty Eppendorf tubes and
was evaporated to dryness in a steam bath until there were no more traces of ethyl acetate
detected. These replicates were then reconstituted with 500 µL of 1.0 M NaCl and the
absorbance of the resulting solution was read at 228 nm using a UV-Vis spectrophotometer.
The percent (%) ACE inhibition of the samples was calculated using the formula:

C − A
 
% ACE Inhibition = × 100 (1)
C − B

where C is the absorbance of the main control, A is the absorbance of the sample or the
positive control, and B is the absorbance of the method blank.

2.9. Determination of the Radical Scavenging Activity by 2.2-Diphenyl-1-picrylhydrazil

(DPPH) Assay
A 2.2-diphenyl-1-picrylhydrazyl (DPPH) assay was carried out according to the
method of Pownall et al. [23] with few modifications. For the sample replicates, 150 µL of
sample (undigested and digested crude, partially purified and purified protein extract)
was placed in each well of 300 µL capacity microtiter plate, followed by the addition of
50 µL of 50 µg/mL DPPH in methanol. For the positive control, 150 µL of 50 µg/mL
ascorbic acid solution in extraction buffer was added instead of samples, while for the main
control, 150 µL methanol was added. The resulting replicate mixtures were incubated for
30 min in the dark. After incubation, the absorbance of the replicates was read at 517 nm
Appl. Sci. 2021, 11, 2190 5 of 13

using a UV-Vis spectrophotometer. The percent (%) scavenging activity of the samples was
calculated using the formula:

Acontrol − Asample
 
% Scavenging Activity = × 100 (2)
Acontrol

where Acontrol corresponds to the absorbance of the main control while Asample corresponds
to the absorbance of the sample.

2.10. Statistical Analysis

The activities were analyzed using GraphPad Prism (GraphPad Software, San Diego,
CA, USA). The activities of the samples were compared to the activity of the corresponding
standard used for each assay using one-way ANOVA and Tukey’s multiple comparisons
test to determine whether these activities are significantly different with respect to the
activity of the standard.

3. Results and Discussion

3.1. Extraction of Proteins from ‘Señorita’ Banana Pulp
This study extracted proteins from ripe ‘Señorita’ banana pulp to determine protein
related
Appl. Sci. 2020, 10, x FOR PEER health
beneficial effects associated to consumption of the fruit, although
REVIEW 6 of 13other plant
parts such as root [24] and peel [25] are also known to contain extractable proteins. The
SDS-PAGE
Densitometric analysis profile
allowed (Figure 1,oflane
estimation the b) of the crude
molecular protein
weights andextract is similar
calculation to the profile
of the
percent band volumeobtained
of each by Surabhi
protein et al. in
present [26]the
with both profiles
SDS-PAGE showing
profiles of themajor
crude,proteins with MW of
partially
purified, and purified approximately
protein extracts.15, 30 showed
This and 40 kDa. This
that the proves
major that proteins
proteins have MW were successfully
of 15 kDa, since extracted
this protein band had from the powdered
the highest percent pulp.
band volume ranging from 26% to 34% per sample lane.

10

MW

Figure 1. SDS-PAGE Figure 1. SDS-PAGE

profiles profiles banana
for the ‘Señorita’ for the ‘Señorita’ banana(a)protein
protein extract: extract:
protein marker (a)and
protein
theirmarker and their
corresponding molecular weights in kDa; (b) 167 μg/mL crude protein extract; (c) 964 μg/mL partially (c) 964 µg/mL
corresponding molecular weights in kDa; (b) 167 µg/mL crude protein extract;
partially
purified protein extract; andpurified proteinpurified
(d) 152 μg/mL extract; protein
and (d) extract.
152 µg/mL purified proteinwas
The electrophoresis extract.
doneThe
in electrophoresis
was done in a 15% gel and run at 110V for 60 min. Highlighted
a 15% gel and run at 110V for 60 min. Highlighted band: Major proteins/Protein of interest. band: Major proteins/Protein
of interest.
Data from Table 1 shows that there was a significant decrease in protein concentration after
The extracted protein has a concentration of 167.32 µg/mL in terms of extraction
every purification procedure. This or
buffer used means that
334.64 non-major
µg/g proteins
in terms of fruitwere
pulpsubsequently removed
used. The low from
concentration of the
the crude and partially purified extract leaving only the major proteins in the purified extract.
protein extracted can be associated to the initially low protein content of the sample,
Although the results of the colorimetric Bradford assay in Table 1 showed a decrease in concentration
after every purification step, the SDS-PAGE profile of partially purified (lane c) and purified extract
(lane d) in Figure 1 had more intense bands compared to the crude extract (lane b). This is due to
loading of preconcentrated partially purified and purified extract into the gel system. If
Appl. Sci. 2021, 11, 2190 6 of 13

difference in the actual protein content of the sample compared to the representative
species reported in the literature, and difference or modification in the implemented
extraction procedure. For example, although the extraction buffer used in the study is
almost the same with the extraction buffer used in in the reference studies [24,25], the
implemented extraction procedure was simplified resulting to a decreased amount of
protein extracted. Extraction procedures on the reference studies have coupled extraction
with different protein precipitation methods and/or have added other components to the
buffer to increase extraction efficiency and yield a protein with higher purity.
Throughout the experiment, pH and temperature were carefully observed since pro-
teins are sensitive to changes in these parameters. Drastic changes in these parameters can
lead to protein denaturation, affecting negatively the efficiency of the protein extraction
procedure and the quality of the protein extracted. Low temperature condition was em-
ployed to prevent any endogenous protease activity which may lead to denaturation of the
protein of interest [4].

3.2. Purification of the Crude Protein Extract

Partial purification of the crude extract using ammonium sulfate precipitation re-
moved proteins that did not precipitate at 40–60% salt saturation. This also concentrated
the dilute crude extract, shown by more intense protein bands (Figure 1, lane c), through
the dissolution of the precipitated proteins in a smaller buffer volume [4]. Dialysis, on
the other hand, removed most of the ammonium sulfate present in the partially purified
sample which could affect the results of the succeeding protein characterization procedures.
Lastly, gel filtration chromatography (Figure 1, lane d) as a purification procedure effec-
tively separated substances with MW range between 10 kDa to 100 kDa which refers to the
effective separative capacity of the resin used.
Comparison of the protein profiles in Figure 1 (lanes b, c and d) showed that the
purification scheme was effective. Protein band at 10 kDa and other faint protein bands at
25–37 kDa area of the crude profile were not observed in the purified profile. Moreover,
the protein band at 15 kDa became more distinct after purification.
Densitometric analysis allowed estimation of the molecular weights and calculation
of the percent band volume of each protein present in the SDS-PAGE profiles of the crude,
partially purified, and purified protein extracts. This showed that the major proteins have
MW of 15 kDa, since this protein band had the highest percent band volume ranging from
26% to 34% per sample lane.
Data from Table 1 shows that there was a significant decrease in protein concentration
after every purification procedure. This means that non-major proteins were subsequently
removed from the crude and partially purified extract leaving only the major proteins in
the purified extract. Although the results of the colorimetric Bradford assay in Table 1
showed a decrease in concentration after every purification step, the SDS-PAGE profile
of partially purified (lane c) and purified extract (lane d) in Figure 1 had more intense
bands compared to the crude extract (lane b). This is due to loading of preconcentrated
partially purified and purified extract into the gel system. If preconcentration of the said
extract were not done, their dilute concentration might result to very faint protein bands.
Meanwhile, the crude extract was not pre-concentrated prior to SDS-PAGE since it already
gave a workable protein profile band with the concentration as it is.
Appl. Sci. 2021, 11, 2190 7 of 13

Table 1. Protein content (µg/g fruit pulp) of the crude, partially purified and purified extracts as
calculated using colorimetric Bradford assay.

Protein Concentration *, Protein Concentration *,

Sample
µg/mL Buffer µg/g Fruit Pulp
Crude Extract 167.32 ± 10.90 a 334.64 ± 21.70 a
Partially Purified Extract 120.45 ± 1.59 b 289.08 ± 3.82 b
Purified Extract 28.51 ± 1.63 c 57.02 ± 3.25 c
* The mean ± SD values of three determinations in the column if followed by the same superscript letters are not
significantly different at 5% level of significance using ordinary one-way ANOVA followed by Tukey’s test.

Appl. Sci. 2020, 10, x FOR PEER Expounding

REVIEW on the final purification procedure, Gel Filtration Chomatography7 of 13 (GFC),
Figure 2 shows that there was only one observable curve which peaked at fractions 20 and
profile showing a curve or band
21. An rather
elution than
profile being narrowly
showing a curve orpeaked suggests
band rather thanthat thenarrowly
being purified protein
peaked suggests
was distributed in the that the purified
fractions foundprotein wascurve
under the distributed
rather in theon
than fractions
a singlefound under
fraction. the curve rather
SDS-PAGE
than
result of these fractions on a single
showed fraction.
the same profileSDS-PAGE result
with protein bandsof becoming
these fractions showedfrom
more intense the same
the profile
with protein
19th up to the 23rd fractions (referbands becoming more
to Supplementary intense from
materials). the 19th up
The fractions to thethe
outside 23rd fractions
curve have (refer to
Supplementary
no visible protein bands. Materials). The fractions outside the curve have no visible protein bands.

Figure 2. Figure
Elution2.profile
Elutionfor
profile
the GFCfor the GFC fractions
fractions of ‘Señorita’
of ‘Señorita’ bananabanana
proteinprotein partially
partially purified
purified at 40%–
at 40–60% ammonium
60% ammonium
sulfate saturation. sulfate saturation.

Reconsidering Figure 1, the distinctFigure

Reconsidering protein band
1, the foundprotein
distinct at 10 kDa
band offound
the crude
at 10and
kDapartially
of the crude and
purified protein extracts did not
partially appear
purified at the extracts
protein purified did
extract. Moreover,
not appear thepurified
at the said protein bandMoreover,
extract. did the
said protein band did not also appear at any GFC fraction even though
not also appear at any GFC fraction even though there were already 60 fractions collected. This there were already
protein band did not60 fractions
elute collected.
out of the column This
as theprotein band
resin only did not separated
effectively elute out proteins
of the column
with MW as the resin
between 10 kDa and only effectively
100 kDa. Proteinsseparated proteinsweight
with molecular with MW between
smaller than 10
thekDa and
given 100 kDa.
range would Proteins
be with
molecular weight smaller than the given range would be trapped and totally
trapped and totally included in the pores of the gel beads with the possibility of not being eluted out included in
the pores of the
by merely using the same elution buffer.gel beads with the possibility of not being eluted out by merely using the
same elution buffer.
3.3. Enzymatic Hydrolysis of the Isolated
3.3. Enzymatic Proteinsof the Isolated Proteins
Hydrolysis
There are known isolated
Thereproteins,
are knownthatisolated
even inproteins,
their intact form,
that evencan exhibit
in their biologically
intact form, canrelated
exhibit biolog-
activities. However, ically related
protein activities.
consumed However,
undergo protein
digestion consumed
before undergo
it can be digestion before
fully metabolized and it can be
fully
utilized by the body as metabolized
source of energyand
andutilized
amino by the Moreover,
acids. body as source of energy
studies by Alukoand[7]
amino acids. Moreover,
and Shahidi
studies
and Zhong [8], suggest thatbythe
Aluko [7] and Shahidi
physiological and Zhong
and functional [8], suggest
properties of that the physiological
proteins are from theand func-
encrypted bioactive peptides. Hence, enzymatic hydrolysis of the protein extracts was done to obtain
the target peptides.
The SDS-PAGE profiles of the crude, partially purified and purified extracts showed that the
major protein bands present in the undigested sample disappeared after digestion (refer to
Supplementary Materials). This implies that the proteins in the undigested sample were hydrolyzed
Appl. Sci. 2021, 11, 2190 8 of 13

tional properties of proteins are from the encrypted bioactive peptides. Hence, enzymatic
hydrolysis of the protein extracts was done to obtain the target peptides.
The SDS-PAGE profiles of the crude, partially purified and purified extracts showed
that the major protein bands present in the undigested sample disappeared after digestion
(refer to Supplementary Materials). This implies that the proteins in the undigested sample
were hydrolyzed up to a certain extent resulting to smaller peptide fragments which
were not resolved during gel electrophoresis. No significant thick band appeared at the
bottom of the electrophoretogram (refer to Supplementary Materials) which should have
resulted from the accumulation of low MW proteins and/or peptides suggesting that these
fragments could have directly run out of the gel system during the electrophoretic run.
Densitometric analysis, used to determine the extent of protein hydrolysis through
the reduction in peak height and area of each observable protein bands, showed that
there were no significant peaks in the 3 h, 4 h, 12 h and 24 h sample digests proving
that the proteins were successfully hydrolyzed into smaller peptide fragments (refer to
Appl. Sci. 2020, 10, x FOR Supplementary
PEER REVIEW Materials). 8 of 13

3.4. In Silico Analysis3.4. InIsolated

of the Silico Analysis of the Isolated
Major Protein Major Protein
from ‘Señorita’ Bananafrom ‘Señorita’ Banana Extracts
Extracts
UniProt KB [20] Protein Search query returned more than 2000 Musa acuminata proteins
UniProt KB [20] Protein Search query returned more than 2000 Musa acuminata proteins which
which have a MW of 15 kDa, the approximate MW of the major protein. Some of the
have a MW of 15 kDa, the approximate MW of the major protein. Some of the proteins returned in
proteins returned in the query includes profilin, histone, thioredoxin, ferredoxin, ribosomal
the query includes profilin,
proteins,histone, thioredoxin,
lectin and ferredoxin,
various enzymes ribosomal
such proteins,
as kinases, lyaseslectin and various
and chitinase. Lectin was
enzymes such as kinases,
used as the putative protein for its sugar binding property and is found its
lyases and chitinase. Lectin was used as the putative protein for sugarinter- and
in both
binding property and is found in
intracellular bothallowing
space inter- and
themintracellular
to be easily space allowing
extracted them totobe
compared easily
other proteins in
extracted compared the to other proteins
search queryin the search
which can bequery
found which cancellular
in the be found in the cellular
organelles organelles
requiring a complicated
requiring a complicated extraction
extraction procedure.
procedure. A study
A study by Al-Alwani
by Al-Alwani [27] showed
[27] showed that lectin
that lectin is easily
is easily extracted from
extracted from whitewhitekidney beansbeans
kidney by using 0.15 M
by using NaCl.
0.15 M NaCl.
Figureprotein
Figure 3 shows the lectin 3 shows the lectinafter
sequence protein sequence
hydrolysis afterthe
using hydrolysis
enzymesusing the trypsin,
pepsin, enzymes pepsin,
chymotrypsin, and trypsin, chymotrypsin,
thermolysin obtained and thermolysin
through obtained [21]
BIOPEP-UWM through BIOPEP-UWM
protein analyzer. The [21] protein
highlighted peptide fragments are known to exhibit ACE inhibitory activity and/or antioxidant activity
analyzer. The highlighted peptide fragments are known to exhibit ACE inhibitory
activity. and/or antioxidant activity.

M - N - G - A - IK - VG - AW - GGN - GGS - A - F - DM - GP - AH - R - I - IS -
VK - I - Y - SGD - V - VDG - VD - VT - F - TS - Y - EK - TETR - H - F -
GGSGGTPH - E - I - V - L - QEGE - Y - L - VGM - TGE - F - AN - Y - H - G - V
- V - V - VGK - L - G - F - N - TN - K - K - S - Y - GP - F - GN - TGGTP - F - S -
L - P - I - V - AGK - ISG - F - F - GR - GGQ - F - L - D - A - IG - V - Y - L - EP

Figure 3. Representation of the cleavage

Figure 3. Representation of the sites (-) ofsites
cleavage pepsin,
(-) oftrypsin,
pepsin,chymotrypsin and thermolysin
trypsin, chymotrypsin in the lectin
and thermolysin in protein
sequence with the peptides
the lectin highlighted
protein sequence arethe
with known to exhibit
peptides ACE inhibitory
highlighted are knownactivity and/or
to exhibit antioxidant
ACE inhibitory activity. Yellow
activity
highlight for ACEantioxidant
and/or inhibitor peptides and
activity. blue highlight for
Yellow for both
ACEACE inhibitor
inhibitor and antioxidant
peptides peptides.
and blue highlight for both
ACE inhibitor and antioxidant peptides.
3.5. Determination of the Percent (%) Angiotensin Converting Enzyme (ACE) Inhibition as a
Basis of Antihypertensive Activity
3.5. Determination of the Percent (%) Angiotensin Converting Enzyme (ACE) Inhibition as a Basis of
Antihypertensive Activity The results in Table 2 confirmed that the sample extracts have ACE inhibitory activity
even though some values were small. The percent ACE inhibition of the samples were
The results in Table 2 confirmed
all significantly that the
different to sample extracts have
that of captopril ACE inhibitory
(standard), even though activity even
the concentration
though some valuesofwere small.was
the drug Theleveled
percenttoACEthe inhibition of the
concentration of samples were(50
the samples all µg/mL).
significantly
A study by
Rhajbar (standard),
different to that of captopril et al. [28] showed that the
even though activity
the of ACE of
concentration inhibitory
the drugcompounds
was leveleddepends
to the on the
concentration of thedosage
samples and
(50identity
μg/mL). ofA
the compound.
study by RhajbarHence, it could
et al. be that the
[28] showed thatlowtheconcentration
activity of of the
samples during the testing have led to the observed low activities.
ACE inhibitory compounds depends on the dosage and identity of the compound. Hence, it could be
that the low concentration of the samples during the testing have led to the observed low activities.
Among the samples tested, the undigested crude protein extract showed the highest activity
with 85.20% while the remaining samples have roughly 10% to 40%. The undigested crude extract
was composed of various proteins in their native conformation as well as traces of other easily
extractable compounds such as polyphenols, which could account for the observed high activity. For
instance, a study by Geng et al. [29] and Lau et al. [30], showed that crude protein extracts from
various mushroom samples inhibited ACE by up to 90% depending on the kind of mushroom and
Appl. Sci. 2021, 11, 2190 9 of 13

Table 2. ACE inhibition (%) of crude, partially purified and purified protein extracts at 0, 3, 4, 12, and 24 h of digestion *.

ACE Inhibition (%)

Digestion Hours
Captopril Crude Partially Purified Purified
0 99.72 ± 0.71 a 85.20 ± 6.80 b,b 21.78 ± 0.37 b,e,f,c 21.74 ± 2.69 b,c
3 − 28.03 ± 3.63 c,b 11.30 ± 0.67 c,c 29.14 ± 0.99 c,b
4 − 33.60 ± 4.75 c,d,b 15.89 ± 1.54 d,e,c 31.25 ± 0.91 c,d,b
12 − 33.73 ± 2.79 c,d,b 18.86 ± 1.64 e,f,c 34.39 ± 0.11 d,e,b
24 − 41.07 ± 2.25 d,b 22.06 ± 2.24 f,c 36.73 ± 2.35 e,b
* The mean ± SD values of three determinations in the column if followed by the same superscript letters are not significantly different at
5% level of significance using ordinary one-way ANOVA followed by Tukey’s test. The letters before the comma shows Tukey’s multiple
comparison result in the given column while the letters after the comma shows Tukey’s multiple comparison result in the given row.

Among the samples tested, the undigested crude protein extract showed the highest
activity with 85.20% while the remaining samples have roughly 10% to 40%. The undigested
crude extract was composed of various proteins in their native conformation as well as
traces of other easily extractable compounds such as polyphenols, which could account
Appl. Sci. 2020, 10, x FOR PEER REVIEW 9 of 13
for the observed high activity. For instance, a study by Geng et al. [29] and Lau et al. [30],
showed that crude protein extracts from various mushroom samples inhibited ACE by up
h and 24-h digests are not significantly different, the number of bioactive peptides released after 12
to 90% depending on the kind of mushroom and extraction methods employed. On the
h of digestion did not significantly increase.
other hand, a study conducted by Rupasinghe [31] showed that flavonoids, a subclass of
polyphenols, extracted from fruit tissues can inhibit ACE with results dependent on the
Table 2. ACE inhibition (%) of crude, partially purified and purified protein extracts at 0, 3, 4, 12, and
sugar moiety attached to the flavonoid ring. Other documented bioactive compounds that
24 h of digestion *.
can inhibit ACE include polysaccharides, sterols, saponins, fiber, vitamins C and E, and
other minerals. ACE Inhibition (%)
Digestion Hours
Statistical
Captopril analysis onCrudethe crude sample at different
Partially Purified digestion hours
Purifiedshowed that there
0 was 99.72
a significant
± 0.71 a decrease
85.20 in the b,b
± 6.80 activity of21.78
undigested
± 0.37 b,e,f,ccrude sample
21.74 ±after
2.69 the
b,c three-hour
3 digestion−followed by28.03 a subsequent
± 3.63 c,b increase11.30
in the activity until 29.14
± 0.67 c,c the 24-h digestion
± 0.99 c,b period
4 (Figure 4). − This implies33.60that thec,d,b
± 4.75 initial hydrolysis of d,e,c
15.89 ± 1.54 the proteins from
31.25 thec,d,bcrude extract
± 0.91
12 led to denaturation
− and
33.73loss of the
± 2.79 c,d,b active native
18.86 ± conformation,
1.64 e,f,c resulting
34.39 ± 0.11tod,e,bthe observed
24 decrease −in activity. On 41.07 2.25 hand, the22.06
the± other d,b ± 2.24 increase in36.73
observed f,c ± 2.35ase,bthe digestion
activity
* The mean ± SD valuesperiodof threeisdeterminations
increased could mean
in the column that the precursor
if followed by the protein(s) were letters
same superscript continuously
are not broken
significantly different atdown to bioactive
5% level peptides
of significance usinginhibiting ACE. However,
ordinary one-way since thebyactivities
ANOVA followed of 12-h
Tukey’s test. The and 24-h
letters before the comma digests
showsare not significantly
Tukey’s different,result
multiple comparison the number of bioactive
in the given column while peptides released
the letters afterafter 12 h
the comma shows Tukey’s of digestion did not significantly
multiple comparison result in theincrease.
given row.

120
85.20

100
ACE Inhibition (%)

80
41.07
33.73
33.60

60 CRUDE
28.03

36.73
34.39
31.25
29.14

Partially Purified
22.06
21.78
21.74

18.86

40
15.89
11.30

Purified
20

0
0 3 4 12 24
Digestion Time, hours

Figure 4. Comparison of ACE inhibition

Figure 4. Comparison (%) of crude,
of ACE inhibition partially
(%) of purified purified
crude, partially and purified protein extracts
and purified at different diges-
protein extracts
tion hours.at different digestion hours.

For the partially purified extract samples, the same trend in the activities of the crude extract
samples was observed. However, the observed activities were found to be the lowest among the three
sample groups. Table 2 shows that there was an observable significant three-fold decrease in the
activity of the undigested crude sample when partially purified. Partial purification removed non-
 Appl. Sci. 2020, 10, x FOR PEER REVIEW 10 of 13

3.6. Determination of the Radical Scavenging Activity by 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Assay as a

Basis of Antioxidant Activity
Appl. Sci. 2021, 11, 2190 10 of 13
For the effect of digestion time on antioxidant activity of the protein extracts, a decrease in the
activity of the partially purified and purified samples was observed as the digestion time was
increased, whereas the activity
For the of the crude
partially samples
purified extract continued
samples, the to increase
same trend up intothe
theactivities
four hour of the crude
digestion time beforeextract
it significantly decreased at the 12 h and 24 h digestion
samples was observed. However, the observed activities were found time (Figure 5).toThis
be the lowest
trend in antioxidantamongactivity,thewhere
three there
sample is groups.
an initial increase
Table 2 showsin activity
that therefollowed
was an by a gradual
observable significant
decline has also beenthree-fold
observed decrease
to other studied [33,34]of the undigested crude sample when partially purified.
in the activity
Table 3 showed Partial
that thepurification
activity of the undigested
removed crude sample
non-protein after three
contaminants andhours and four hours
unprecipitated proteins from
of digestion increased theand
crude extract.
found to beThenot removal of these
significantly components,
different with thewhich
activitymight have ACE
of ascorbic acidinhibitory
(positive control). Thisactivity,
implies decreased significantly
that the initially active the activityfound
proteins of the in
undigested partially
the undigested purified
crude sample sample. On
released peptides with the other
high hand, the increase
antioxidant in the
activity activity of
However, the purified
further sample
hydrolysis of could
thesebe associated to the
peptides
decreased the activitypeptides inhibiting
of the sample the ACEintothe
as observed have more effective
activities of the 12 interaction due to the
h and 24 h digests, decrease in the
showing
sample components that have prevented otherwise
that the peptides with high activity might have been degraded to smaller peptides with lower[32].
activity. For the purified extract samples, there was an increase in activity starting from the
undigested sample up to the 24-h digest. This trend was different to the other two sample
groups. Purified samples were primarily composed of the major protein, as a result, the
Table 3. DPPH radical scavenging activity (%) of crude, partially purified and purified protein
activities observed had only reflected the characteristic activity of the major protein. In
extracts at different digestion hours *.
silico analysis of the putative protein showed that enzymatic hydrolysis of the protein
will release bioactive peptides that can
DPPH Radical inhibit ACE,
Scavenging thus,
Activity (%) prolonging the hydrolysis of the
Digestion Hours
protein through
Ascorbic Acidincreased Crude
digestion periodPartially
released more bioactivePurified
Purified peptides resulting to
0 an observable
36.31 ± 0.21increase
a
in activity.
17.21 ± 0.27 b,b 18.00 ± 0.72 b,b 17.37 ± 1.69 b,b
3 − 30.83 ± 1.36 a,b 14.55 ±1.24 c,c 16.59 ± 1.65 b,c
4 3.6. Determination
− of the 34.74
Radical Scavenging
± 1.24 a,a Activity
13.30 ±by 2,2-Diphenyl-1-picrylhydrazyl
0.98 c,b 14.87 ± 1.18 b,b (DPPH)
12 Assay as a −Basis of Antioxidant
12.52 ±Activity
1.18 b,b 15.02 ± 0.47 c,c 6.73 ± 0.54 c,d
For the
− effect of digestion
24 −107.36 ±time
5.15on
c,b antioxidant
1.41 ±activity
1.69 d,c of the protein
1.41 ± extracts,
2.93 d,c a decrease
in the activity of the partially purified and purified samples was
* The mean ± SD values of three determinations in the column if followed by the same superscriptobserved as the digestion
time was increased,
letters are not significantly different atwhereas
5% level the activity of the
of significance crude
using samples
ordinary continued
one-way ANOVA to increase up to
followed by Tukey’sthetest.
fourThe
hour digestion
letters before time beforeshows
the comma it significantly decreased
Tukey’s multiple at the 12result
comparison h and in 24 h digestion
time (Figure 5). This trend in antioxidant activity, where there
the given column while the letters after the comma shows Tukey’s multiple comparison result in theis an initial increase in
given row. activity followed by a gradual decline has also been observed to other studied [33,34]

50
34.74
30.83
Radical Scavenging Activity (%)

40
17.21
18.00
17.37

30
16.59

12.52
15.02
14.55

14.87
13.30

Crude
20
Partially Purified
6.73

0.00
1.41
1.41

10 Purified

0
0 3 4 12 24
-10
Digestion Time, hours

Figure 5. Comparison of DPPH radical scavenging activity (RSA) of crude, partially purified and purified protein extracts
Figure 5. Comparison of DPPH radical scavenging activity (RSA) of crude, partially purified and
at different digestion hours. (Note: DPPH RSA of crude at 24 h digestion time is not included due to highly negative value
purified protein extracts at different digestion hours. (Note: DPPH RSA of crude at 24 h digestion
obtained, −107.355%).
time is not included due to highly negative value obtained, −107.355%).
Table 3 showed that the activity of the undigested crude sample after three hours
On the other hand,
andthe observed
four hours ofgeneral decrease
digestion in activity
increased of theto
and found partially purified and purified
be not significantly different with the
protein samples as the digestion period was increased, could mean that during hydrolysis, the proteins
activity of ascorbic acid (positive control). This implies that the initially active
initially active proteins present in the undigested samples did not release any peptides with high
found in the undigested crude sample released peptides with high antioxidant activity
However, further hydrolysis of these peptides decreased the activity of the sample as
observed in the activities of the 12 h and 24 h digests, showing that the peptides with high
activity might have been degraded to smaller peptides with lower activity.
Appl. Sci. 2021, 11, 2190 11 of 13

Table 3. DPPH radical scavenging activity (%) of crude, partially purified and purified protein extracts at different digestion
hours *.

DPPH Radical Scavenging Activity (%)

Digestion Hours
Ascorbic Acid Crude Partially Purified Purified
0 36.31 ± 0.21 a 17.21 ± 0.27 b,b 18.00 ± 0.72 b,b 17.37 ± 1.69 b,b
3 − 30.83 ± 1.36 a,b 14.55 ±1.24 c,c 16.59 ± 1.65 b,c
4 − 34.74 ± 1.24 a,a 13.30 ± 0.98 c,b 14.87 ± 1.18 b,b
12 − 12.52 ± 1.18 b,b 15.02 ± 0.47 c,c 6.73 ± 0.54 c,d
24 − −107.36 ± 5.15 c,b 1.41 ± 1.69 d,c 1.41 ± 2.93 d,c
* The mean ± SD values of three determinations in the column if followed by the same superscript letters are not significantly different at
5% level of significance using ordinary one-way ANOVA followed by Tukey’s test. The letters before the comma shows Tukey’s multiple
comparison result in the given column while the letters after the comma shows Tukey’s multiple comparison result in the given row.

On the other hand, the observed general decrease in activity of the partially purified
and purified protein samples as the digestion period was increased, could mean that during
hydrolysis, the initially active proteins present in the undigested samples did not release
any peptides with high antioxidant activity, rather they were continuously degraded to
peptides with lower activity. This observation is contrary to the result of in silico analysis.
Statistical analysis of the crude, partially purified, and purified sample activities
at a specific digestion period showed that the activities of undigested samples where
not significantly different from each other, whereas, for most digested samples, only the
activity from the digested crude samples were found to be significantly different. For the
undigested samples, it could be that the activities observed were solely from the extracted
and purified major protein, hence, the activities were not significantly different from
each other. Meanwhile, the observed significantly different activity of the crude digested
samples compared to other digested samples could be the result of the activity of peptides
released from the non-major proteins removed during purification. In silico analysis of
the putative major protein (Figure 3) showed that only few antioxidant peptides will be
released upon hydrolysis justifying the low activities of the digested partially purified and
purified samples

4. Conclusions
The study extracted, purified and hydrolyzed protein from ‘Señorita’ banana pulp
with the major protein having an approximate molecular weight of 15 kDa. The low
concentration of the crude extract (167.32 µg/mL buffer) was associated to the characteristic
low protein content of the sample. Furthermore, these extracts and the peptides released
during the enzymatic hydrolysis were tested for ACE inhibitory and antioxidant activities.
The undigested crude sample had the highest ACE inhibitory activity (85.20%), but it was
found to be significantly different from the activity of the standard, captopril (99.72%). On
the other hand, the 3 h and 4 h crude digests had the highest DPPH radical scavenging
activity (30.83% and 34.74%, respectively) that were found to be not significantly different
to the activity of the standard, ascorbic acid (36.31%). For the effect of digestion period,
longer digestion time results to higher ACE inhibitory activity while for the DPPH radical
scavenging activity, there is an initial increase followed by a decline at longer digestion
time. These are expected based on the result of in silico analysis of the putative major
protein, lectin, and the available literatures similar to the study. Over-all, this study can
serve as an additional reference to the very limited banana protein and peptide studies.

Supplementary Materials: The Supplementary Materials are available online at https://www.mdpi.

com/2076-3417/11/5/2190/s1.
Author Contributions: Conceptualization, J.M.F., M.A.T., M.C.M.C. and A.H.-F.; formal analysis,
J.M.F.; investigation: J.M.F.; methodology, J.M.F., M.A.T., M.C.M.C. and A.H.-F.; resources, M.A.T.;
supervision, M.A.T.; visualization, J.M.F. and M.A.T.; writing—original draft preparation, J.M.F.,
Appl. Sci. 2021, 11, 2190 12 of 13

M.A.T. and L.Y.U.; writing—review and editing, J.M.F., M.A.T., M.C.M.C., A.H.-F. and L.Y.U. All
authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: The authors acknowledge Process Biochemistry Laboratory (PS B-307)—University
of the Philippines Los Baños and nearby laboratories of the Institute of Chemistry for the support on
this study. The same goes with the Enhanced Creative Work and Research Grant (ECWRG), Office of
the Vice President for Academic Affairs (OVPAA) of the UP System for extending their support to
make this research possible.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. United Nations–Food and Agriculture Organization [UN FAO]. The World Banana Economy 1985–2002. Available online: http:
//www.fao.org/docrep/0.07/y5102e/y5102e00.htm (accessed on 20 November 2017).
2. U.S Department of Agriculture, Agricultural Research Service, Nutrient Data Laboratory. USDA National Nutrient Database for
Standard Reference, Release 27. 2014. Available online: http://www.ars.usda.gov/nutrientdata (accessed on 25 October 2017).
3. Singh, B.; Singh, J.P.; Kaur, A.; Singh, N. Bioactive compounds in banana and their associated health benefits—A review. Food
Chem. 2016, 201, 1–11. [CrossRef] [PubMed]
4. Hames, D.; Hooper, N. Instant Notes in Biochemistry, 3rd ed.; Taylor and Francis Group: London, UK, 2000; ISBN 0-4153-6778-6.
5. Righetti, P.G.; Esteve, C.; D’Amato, A.; Fasoli, E.; Luisa-Marina, M.; Concepcion-Garcia, M. A sarabande of tropical fruit
proteomics: Avocado, banana, and mango. Proteomics 2015, 15, 1639–1645. [CrossRef] [PubMed]
6. Hartmann, R.; Meisel, H. Food-derived peptides with biological activity: From research to food applications. Biotechnology 2007,
18, 163–169. [CrossRef] [PubMed]
7. Aluko, R.E. Determination of nutritional bioactive properties of peptides in enzymatic pea, chickpea and mungbean protein
hydrolysates. J. AOAC Int. 2008, 91, 947–956. [CrossRef]
8. Shahidi, F.; Zhong, Y. Bioactive peptides. J. AOAC Int. 2008, 91, 914–931. [CrossRef]
9. World Health Organization [WHO]. A Global Brief on Hypertension 2012; WHO: Geneva, Switzerland, 2013; Available online:
http://www.who.int/cardiovascular_diseases/publications/global_brief_hypertension/en/ (accessed on 30 November 2017).
10. Philippine Council for Health and Research Development [PCHRD]. Prevalence of Hypertension among Filipinos–increasing
PSH. 28 May 2012. Philippine Council for Health Research and Development Web Site. Available online: http://www.pchrd.dost.
gov.ph/index.php/news/2806-prevalence-of-hypertension-among-filipinos-increasing-psh (accessed on 7 November 2017).
11. Kostis, J.B.; Shelton, B.; Gosselin, G.; Goulet, C.; Hood, W.B., Jr.; Kohn, R.M.; Kubo, S.H.; Shron, E.; Weiss, M.B.; Willis, P.W.; et al.
Adverse effects of enalapril in the studies of left ventricular dysfunction (SOLVD). Am. Heart J. 1996, 131, 350–355. [CrossRef]
12. Mandal, J.; Ponnambath, D.K.; Parija, S.C. Utilitarian and deontological ethics in medicine. Trop. Parasitol. 2016, 6, 5–7. [CrossRef]
13. Mishra, K.; Ojha, H.; Chaudhury, N.K. Estimation of antiradical properties of antioxidants using DPPH assay: A critical review
and results. J. Food Chem. 2012, 130, 1036–1043. [CrossRef]
14. Bondet, V.; Brand-Williams, W.; Berset, C. Kinetics and mechanisms of antioxidant activity using the DPPH free radical method.
Lebensm. Wiss. Technol. 1997, 30, 609–615. [CrossRef]
15. Sakanaka, S.; Tachibana, Y. Active oxygen scavenging activity of egg-yolk protein hydrolysates and their effects on lipid oxidation
in beef and tuna homogenates. Food Chem. 2006, 95, 243–249. [CrossRef]
16. Weiss, J.R.; Landauer, M.R. Protection against ionizing radiation by antioxidant nutrients and phytochemicals. Toxicology 2003,
189, 1–20. [CrossRef]
17. Esteve, C.; D’Amanto, A.; Garcia, M.C.; Righetti, P.G. In-Depth Proteomic Analysis of Banana (Musa spp.) Fruit with Combinatorial
Peptide Ligand Libraries; Department of Analytical Chemistry, Miles Gloriusus Academy: Milan, Italy, 2012.
18. Bradford, M.M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal. Biochem. 1976, 72, 248–254. [CrossRef]
19. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature 1970, 227, 680–685.
[CrossRef] [PubMed]
20. Uniprot KB. Available online: http://www.uniprot.org/ (accessed on 15 February 2019).
21. BIOPEP–UWM. Available online: http://www.uwm.edu.pl/biochemia/index.php/en/biopep (accessed on 15 February 2019).
22. Cushman, D.W.; Cheung, S.H.; Sabo, E.F.; Ondetti, M.A. Spectrophotometric assay and properties of the angiotensin-I converting
enzyme of rabbit lung. Biochem. Pharmacol. 1971, 20, 1637–1648. [CrossRef]
23. Pownall, T.L.; Udenigwe, C.C.; Aluko, R.E. Amino acid composition and antioxidant properties of pea seed (Pisum sativum L.)
enzymatic protein hydrolysate fractions. J. Agric. Food Chem. 2010, 58, 4712–4718. [CrossRef] [PubMed]
24. Vaganan, M.M.; Sarumathi, S.; Nandakumar, A.; Ravi, I.; Mustaffa, M.M. Evaluation of protein extraction methods for banana
(Musa spp.) root proteome analysis by two-dimensional electrophoresis. Indian J. Biochem. Biophys. 2014, 52, 101–106.
Appl. Sci. 2021, 11, 2190 13 of 13

25. Zhang, L.L.; Feng, R.J.; Zhang, Y.D. Evaluation of different methods of protein extraction and identification of differentially
expressed proteins upon ethylene-induced early ripening in banana peels. Sci. Food Agric. 2012, 92, 2106–2115. [CrossRef]
[PubMed]
26. Surabhi, G.K.; Pattanaik, S.; Mohanty, S. A comparative method for protein extraction and proteome analysis by two-dimensional
gel electrophoresis from banana fruit. Hortic. Biotechnol. Res. 2016, 2, 8–13. [CrossRef]
27. Al-Alwani, B.A.; Jebor, M.A.H.; Jalil, Y. Extraction, purification and characterization of lectin from Phaseolus vulgaris L. cv. white
seeds (white kidney bean). Sci. Bul. Ser. F Biotechnol. 2013, 17, 69–77.
28. Rhajbar, K.; Dawda, H.; Mukundan, U. Polyphenols: Methods of extraction. Sci. Rev. Chem. Commun. 2015, 5, 1–6.
29. Geng, X.; Tian, G.; Zhang, W.; Zhao, Y.; Zhao, L.; Ryu, M.; Wang, H.; Ng, T.B. Isolation of angiotensin I-converting enzyme
inhibitory protein with antihypertensive effect in spontaneously hypertensive rats from the edible wild mushroom Leucopaxillus
tricolor. Molecules 2015, 20, 10141–10153. [CrossRef] [PubMed]
30. Lau, C.C.; Abdullah, N.; Shib, A.S.; Aminudin, N. Proteomic analysis of antihypertensive proteins in edible mushrooms. J. Agric.
Food Chem. 2012, 60, 12341–12348. [CrossRef]
31. Rupasinghe, H.P.V. Inhibition of angiotensin converting enzyme (ACE) by fruit flavonoids in regulation of hypertension. In
Proceedings of the 2nd World Congress on Diabetes & Metabolism, Philadelphia, PA, USA, 6–8 December 2011. [CrossRef]
32. Mahdavi-Yekta, M.; Nouri, L.; Azizi, M.H. The effects of hydrolysis condition on antioxidant activity of protein hydrolyzate from
quinoa. Food Sci. Nutr. 2019, 7, 930–936. [CrossRef] [PubMed]
33. Zhidong, L.; Benheng, G.; Xuezhong, C.; Zhenmin, L.; Yun, D.; Hongliang, H.; Wen, R. Optimisation of hydrolysis conditions for
antioxidant hydrolysate production from whey protein isolates using response surface methodology. Irish J. Agric. Food Res. 2013,
52, 53–65.
34. Bisswanger, H. Review: Enzyme assays. Perspect. Sci. 2014, 1, 41–55. [CrossRef]