CyTA - Journal of Food

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Antioxidant, angiotensin-converting enzyme,

and α-amylase inhibitory activities of protein
hydrolysates of Leucaena leucocephala seeds

Iván Balderas-León, Diana Baigts-Allende & Anaberta Cardador-Martínez

To cite this article: Iván Balderas-León, Diana Baigts-Allende & Anaberta Cardador-Martínez
(2021) Antioxidant, angiotensin-converting enzyme, and α-amylase inhibitory activities of protein
hydrolysates of Leucaena�leucocephala seeds, CyTA - Journal of Food, 19:1, 349-359, DOI:
10.1080/19476337.2021.1909144

To link to this article: https://doi.org/10.1080/19476337.2021.1909144

© 2021 The Author(s). Published with

license by Taylor & Francis Group, LLC.

Published online: 20 Apr 2021.

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CYTA – JOURNAL OF FOOD
2021, VOL. 19, NO. 1, 349–359
https://doi.org/10.1080/19476337.2021.1909144

Antioxidant, angiotensin-converting enzyme, and α-amylase inhibitory activities

of protein hydrolysates of Leucaena leucocephala seeds
a a,b a
Iván Balderas-León , Diana Baigts-Allende and Anaberta Cardador-Martínez
a
Tecnologico de Monterrey, Escuela de Ingeniería y Ciencias, Querétaro, México; bDepartamento de Ingeniería Química, Ambiental y de
Alimentos, Universidad de las Américas, Puebla, México

ABSTRACT ARTICLE HISTORY

The Leucaena leucocephala seeds (LLS) cotyledon proteins, globulins, and glutelins were indepen­ Received 2 December 2020
dently hydrolyzed by Alcalase, Trypsin, and α-Chymotrypsin up to 180 min. The degree of hydrolysis Accepted 22 March 2021
(DH), antioxidant, ACE-inhibitory, and α-Amylase inhibition were assessed. The higher DH values KEYWORDS
were 76.75% and 82.71% for globulin α-Chymotrypsin, and glutelin Alcalase hydrolysates, respec­ Antioxidant activity; protein
tively. The glutelin hydrolyzed with Trypsin showed the higher DPPH• antioxidant activity while the hydrolysate; bioactive
higher ABTS antioxidant activity was for globulin Alcalase hydrolysate after 180 min digestion. The peptides; nutraceutical; ACE
highest ACE-inhibitory activity was 95.44% for globulin-Alcalase hydrolysate at 180 min; while, for inhibition
the α-Amylase inhibition assay, glutelin-Alcalase showed the highest values at 100 min of reaction.
LLS cotyledon protein concentrates and hydrolysates with relatively low mimosine content were PALABRAS CLAVE
actividad antioxidante;
obtained after protein extraction. Such findings indicate the possibility of getting bioactive peptides hidrolizados proteicos;
from LLS cotyledon proteins using enzyme digestions and might be utilized for functional foods péptidos bioactivos;
with physiological enhancer effects. nutraceúticos; Inhibición de
ACE
RESUMEN
El concentrado proteico y las fracciones de globulinas y glutelinas del cotiledón de semillas de
Leucaena leucocephala (LLS) fueron hidrolizados con Alcalasa, Tripsina y α-quimotripsina hasta 180
min. El grado de hidrólisis (DH), actividades antioxidante e inhibitoria de las enzimas convertidora
de angiotensina (ACE-I) y α-amilasa fueron medidas. Los grados de hidrólisis más altos fueron
76.75% y 82.71% para los hidrolizados de globulina-α quimotripsina y glutelina-Alcalasa, respecti­
vamente. El hidrolizado de glutelina con Tripsina mostró la mayor actividad antioxidante con DPPH,
mientras que el hidrolizado de globulina con alcalasa fue el más alto a los 180 min en el método
ABTS. La mayor actividad de ACE-I (95.44%) se observó en el hidrolizado de globulinas-Alcalasa. Por
el contrario, el hidrolizado de glutelinas-Alcalasa a los 100 min mostró la mayor actividad. Se
obtuvieron concentrados proteicos e hidrolizados con un contenido de mimosina relativamente
bajo. Estos resultados indican la posibilidad de obtener péptidos bioactivos a partir del cotiledón de
LLS usando digestión enzimática los cuales podrían utilizarse en alimentos funcionales con efectos
fisiológicos.

Introduction used to treat hypertension in humans. However, synthetic

In the last decades, the degenerative disease rate has increased
due to lifestyle changes (Delles et al., 2018). Nowadays, dis­
eases such as hypertension and other cardiovascular are the
leading cause of morbidity and mortality in Western countries.
About 20% of the adult population suffers from hypertension
(Fan et al., 2019). Moreover, CDC (2020) reported a prevalence
of 45% hypertensive adults in the United States. The control of
blood pressure is mediated by various regulatory mechanisms
in the body, including the angiotensin I-converting enzyme
(ACE-I) (Wu et al., 2006). This enzyme produces angiotensin II,
using angiotensin I as substrate. The final product of ACE-I is
the octapeptide Angiotensin II, which causes peripheral vaso­
constriction, and sodium reabsorption in the kidney while
increasing body fluids.
Additionally, angiotensin III, produced from angiotensin II,
controls the production of aldosterone and raises blood
pressure (Martinez-Maqueda et al., 2012). The discovery of
bioactive peptides in snake venom led to the synthesis of
ACE-I inhibitors such as captopril and enalapril, currently
drugs could produce side effects such as cough, taste altera­
tions, and skin rashes. Therefore, natural ACE-I inhibitors’
interest has increased (Daskaya-Dikmen et al., 2017).
On the other hand, reactive oxygen species (ROS) such as
the superoxide radical, hydrogen peroxide, and hydroxyl
radicals are physiological metabolites formed due to aerobic
organisms’ respiration (Siow & Gan, 2013; Yea et al., 2014).
ROS are unstable and can react rapidly with membrane
lipids, proteins, and DNA, resulting in cell damage
(Rudolph et al., 2017). The imbalance between oxidizing
species and endogenous antioxidants causes oxidative
stress. Moreover, oxidative stress has been associated with
degenerative diseases like cardiovascular disorders and type
II diabetes (Martinez-Leo et al., 2019). The intake of antiox­
idants could reduce the morbidity and mortality associated
with coronary heart diseases, as demonstrated in several
studies (Durak et al., 2013). Consequently, the search for
food antioxidants that could protect the body from ROS
and retard many chronic diseases’ evolution is steadily
increasing (López-Barrios et al., 2014).

CONTACT Anaberta Cardador-Martínez mcardador@tec.mx Tecnologico de Monterrey, Escuela de Ingeniería y Ciencias, Epigmenio González 500, San
Pablo, Querétaro 76130, México.
© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which
permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
350 I. BALDERAS-LEÓN ET AL.
Studies on foods as natural sources of ACE-I inhibitors and
antioxidants fall clearly within the scope of functional foods
research. ACE-I inhibitory and antioxidant peptides derived
from food proteins are excellent potential ingredients in func­
tional foods (Torruco-Uco et al., 2009). These protein fragments
may be released from the parent protein in vivo (i.e. during
gastrointestinal digestion) or in vitro (i.e. during food proces­
sing). Bioactive peptides with ACE-I α-amylase-inhibitory and
antioxidant activities have been produced through commer­
cial enzyme digestion (Barbana & Boye, 2010).
The Guaje (Leucaena leucocephala) is a legume crop
grown predominantly in Mexico’s tropical and subtropical
areas. L. leucocephala seeds are currently used as a dietary
protein source in both human and animal diets. However,
they could be an excellent raw material of protein concen­
trates (Zárate et al., 2005). L. leucocephala is not recom­
mended for extensive human consumption due to
mimosine. Mimosine is an alkaloid that causes hair loss,
growth retardation, cataract, goiter, and decreased fertility,
and mortality in non-ruminant mammals. Furthermore,
L. leucocephala seed protein isolates with relatively low
mimosine levels have been prepared successfully by isoelec­
tric precipitation of seed kernel proteins; it has been used to
overcome mimosine toxicity since mimosine was left in the
supernatant (Sethi & Kulkarni, 1993).
Protein extracts have been used to produce hydrolysates
with improved functionality and/or nutritional properties
(López-Barrios et al., 2014). Vegetable proteins are exciting,
and legumes are especially promising because of their high
protein content and diverse physiological activities in
humans, for instance, antihypertensive and antioxidant
effects (Lammi et al., 2019; Rudolph et al., 2017). Guaje
protein hydrolysates may also represent a source of bioac­
tive peptides with potential applications in developing novel
physiologically functional foods, enhancing the value of the
hydrolysates and overall value of the starting material, i.e.
protein extracts.
This work’s objective was to determine in vitro antioxi­
dant, ABTS•+ and DPPH• scavenging activities, ACE-I, and α-
Amylase inhibitory properties for Leucaena leucocephala
seeds (LLS) cotyledon protein hydrolyzed with Alcalase,
Trypsin, and α-Chymotrypsin. Additionally, the mimosine
reduction during the preparation of protein concentrates
was evaluated.
Materials and methods
ACE (EC: 3.4.15.1, from porcine kidney, 0.5 U), α-Amylase (EC:
3.2.1.1, from porcine pancreas, 2 U), Alcalase® (EC: 3.4.21.62,
from Bacillus licheniformis, ≥2.4 U/g protein), Trypsin® (EC:
3.4.21.4 from bovine pancreas, 2000 U/mg protein),
α-Chymotrypsin® (EC: 3.4.21.1 from bovine pancreas,
≥40 U/mg protein) and mimosine, were purchased from
Sigma, St. Louis, MO, USA while HPLC-grade acetonitrile
was purchased from Fisher Scientific, Nepean, ON, Canada.
All other chemicals were reagent and obtained from Sigma
or Fisher Scientific. HPLC-grade water was generated by
a Milli-Q system (Millipore, Bedford, MA, USA).
Raw material preparation
The pods of L. leucocephala, without visible morphological
alterations, were obtained at a local market in Oaxaca,
Mexico. First, the seeds were separated from the pod and
soaked in distilled water for 2 h. Afterward, seeds were
manually dehulled, and the cotyledon obtained was air-
dried at 60°C during 48 h (BinderTM ED 23, Germany), ground
in an electric miller (KrupsTM GX4100, Germany) and sieved
through a 625 μm mesh. Finally, LLS flour was kept at −40°C
until usage.
Protein concentrates preparation
Globulins and glutelins protein concentrates were obtained
by isoelectric precipitation following the method of
Rodriguez-Ambriz et al. (2005). Briefly, to generate globulins
concentrates, the LLS cotyledon flour was mixed with NaCl
5% solution in a 1:10 (w/v) ratio and then stirred for 2 h at
room temperature with mechanical agitation. After this time,
the suspension was centrifuged at 5000 x g for 15 min at 4°C
(Thermo Scientific SorvallTM ST 16). After that, the super­
natant was collected, and the extractions were repeated
twice on the remaining seed flour at the same flour/solvent
ratio. For glutelins extraction, the LLS cotyledon flour was
suspended in water (10% w/v), and the pH was adjusted to
9, adding 1 N NaOH, using the same conditions previously
described. The supernatant of each extract, collected sepa­
rately, was joined, and the protein from both supernatants
was acid precipitated (pH 6) with 0.1 N HCl and recovered by
centrifugation (5000 x g, 15 min, 4°C). The supernatants were
discarded. Afterward, each protein pellet was washed twice
with distilled water, centrifuged (5000 x g for 15 min at 4°C),
and immediately lyophilized (Labconco™ Triad 740040,
USA). Once lyophilized, globulins and glutelins concentrates
were stored at −40°C until use.
Protein hydrolysis
Globulin and glutelin enzymatic hydrolysis were carried out
according to Boschin et al. (2014). Three independent
enzymes: Alcalase, Trypsin, and α-Chymotrypsin, were used
in a completely randomized block design. The globulin or
glutelin concentrates were dispersed in water to obtain a 1%
solution (w/v), and the enzyme: substrate ratio was 1:100
(w:v). Hydrolysis with Alcalase was performed at 50°C and pH
8, while hydrolysis with Trypsin and α-Chymotrypsin were
conducted at 37°C and pH 7.8. The pH was kept constant
during the hydrolysis process by adding 0.1 N HCl or 0.1
N NaOH. Aliquots were removed at time intervals from 0,
each 20 min until 180 min, and immediately heated at 95°C
in a water bath for 15 min to stop the enzyme reaction. Then
the hydrolysates were obtained by centrifugation at 10,000 g
for 15 min and stored at −40°C until use.
Degree of hydrolysis measurement
The degree of hydrolysis (DH) was calculated according to
Nielsen et al. (2001) as DH = h/htot × 100, where htot is the
total number of peptide bonds per protein equivalent, and
h is the number of hydrolyzed bonds. The htot factor is
dependent on the amino acid composition of the raw mate­
rial. For LLS, values for α, β, and htot were 1, 0.4, and 8.3,
respectively (Nielsen et al., 2001) for specific raw materials.
Triplicate determinations were performed and measured at
340 nm, using a microplate reader (xMarkTM Microplate

Spectrophotometer). The value of h was calculated using
Equations (1) and (2).
ðserine NH2 βÞ
h¼
α 10 µL. The column was eluted (0.5 mL/min) with two solvent
Serine NH2 ¼ 0:9516 � 0:1 � 100
� �
ODsample ODblank 1 1
� � �
ODstandard ODblank X P
Where, serine-NH2 = meq serine NH2/g protein; X = g sam­
ple; P = protein % in sample; 0.1 is the sample volume.
DPPH• scavenging activity
The DPPH• (2,2-diphenyl-1-picryl- hydrazyl-hydrate) scaven­
ging activity of LL globulins and glutelins hydrolysates was
measured by the method of Bkhairia et al. (2016). Twenty
microliters of the sample were applied to a 96-well plate,
and 200 μL of 125 µM DPPH solution in methanol was
added. The sample’s absorbance was determined at
515 nm after 90 min of incubation. The control was a mixture
of 20 μL distilled water and 200 μL of 125 µM DPPH. The
DPPH scavenging activity was calculated using Equation (3).
� �
Acontrol Asample
DPPH� radical scavenging activityð%Þ ¼
Acontrol
ABTS•+ method
The ABTS•+ (2,2′-azino-bis(3-ethylbenzothiazoline-6- sulfonic
acid) scavenging activity of LLS hydrolysates was determined
by the method of Karamać et al. (2014) carrying out the
reaction on 96 multi-well plates. The ABTS•+ radical was
prepared by the reaction of 5 mL of 7 mM ABTS solution
and 88 μL of 140 mM potassium persulfate solution. The
mixture was kept in the dark at room temperature for 16 h.
After this time, 1 mL of the mixture was diluted with ethanol
to obtain absorbance from 0.70 ± 0.05 at 734 nm. Then,
20 µL portions of the sample were applied onto a 96-well
plate, and 200 µL of ABTS•+ solution was added. The absor­
bance was read after 6 min at 734 nm. Deionized water was
used as control. Percentage inhibition of the ABTS•+ radical
was then calculated using Equation (4).
� �
Acontrol Asample
ABTS�þ radical inhibition percentageð%Þ ¼ �100
Acontrol
Angiotensin I-converting enzyme inhibitory assay
ACE inhibitory activity was performed by the in vitro method
of Wu et al. (2002). ACE 0.1 U/mL and 5 mM HHL (hyppuryl-
histidyl-leucine) were prepared by dissolution in 100 mM
borate buffer (100 mM, pH 8.3) supplemented with
300 mM NaCl. Also, 5 mM of HA (control) and 5 mM
Captopril (standard ACE inhibitor) were dissolved in distilled
water. The reaction system was: 10 µL HHL, 10 µL ACE, 40 µL
ACE inhibitors (hydrolysates), and 40 µL 100 mM borate
buffer (100 mM, pH 8.3). The reaction mixture was incubated
at 37°C for 30 min, and then 250 µL HCl was added.
CYTA - JOURNAL OF FOOD 351
Samples were analyzed using an RP-HPLC in an Agilent
Technologies 1200 series (Palo Alto, CA, USA) equipped with
a Zorbax Eclipse XDB–C18 column (5 µm, 4.6 µm i.d. ×
(1) 150 mm, Agilent, USA) at 25°C. The injection volume was
systems (A) 0.05% TFA in water and (B) acetonitrile (ACN),
with a 5-60% ACN gradient for the first 10 min, maintained
for 2 min at 60% ACN, then returned to 5% ACN for 1 min,
followed by isocratic elution for 4 min. Hypuric acid was
(2)
detected in a diode array detector set a 225 nm. The inhibi­
tory rate was calculated as described by Wu et al. (2002).
α-Amylase inhibitory assay
The determination of α-Amylase inhibitory activity was con­
ducted according to the method described by Siow et al.
(2017). In brief, 100 μL of the sample and 100 μL of sodium
phosphate buffer 20 mM (with NaCl 6 mM, pH 6.9) contain­
ing α-Amylase solution (2 U/mL) were mixed and incubated
for 10 min at 25°C. After this time, 100 μL of starch solution
(1% w/v) in sodium phosphate buffer 20 mM (with NaCl
6 mM, pH 6.9) was then added to start the reaction. The
reaction mixture was incubated at 25°C for 10 min and then
boiled for 5 min in a water bath after adding 200 μL of
dinitrosalicylic acid reagent (DNS) to halt the reaction. The
mixture was cooled to room temperature and diluted with
�100 3 mL of distilled water. The absorbance of the sample, con­
trol 1 (a mixture of starch solution and sample, without the
(3)
addition of the enzyme), and control 2 (a mixture of starch
solution and enzyme, without the addition of the sample)
was measured at 540 nm using a UV-Vis spectrophotometer
(xMarkTM Microplate Spectrophotometer). The α-Amylase
inhibitory activity was expressed as percent inhibition.
Mimosine content
Mimosine content was determined in the LLS cotyledon
flour saline, alkaline extracts, the protein concentrates (glo­
bulins and glutelins), and the 180 min protein hydrolysates
following the protocol of Rodrigues-Corrêa et al. (2019).
Briefly, the dry LLS cotyledon flour (20 mg) was mixed in
a proportion of 1:10 (w/v) with NaCl 5% or NaOH 1 M pH 8;
both mixtures were vortexed for 1 min. All tube samples
were centrifuged at 5000 x g for 15 min at 4°C, and the
supernatants were filtered through a 0.2 μm membrane.
Samples were analyzed in an Agilent Technologies 1200
series with a Zorbax Eclipse XDB–C18 column (5 µm,
4.6 µm i.d. × 150 mm) at 60 . A total of 10 μL of sample
solution was injected into the column and eluted with
(4)
a mobile phase of 0.02 M orthophosphoric acid (a constant
flow of 1 mL/min). Mimosine detection was done at 280 nm,
showing a retention time of 1.48 ± 0.042 min. Mimosine
content in the supernatants was calculated using
a calibration curve (from 0.5-0.0001 mg/mL).
Statistical analysis
Results were expressed as the mean ± standard deviation
(SD) of six determinations. Tukey’s HSD test performed
a comparison of the means at a 5% level of significance.
A Two-way ANOVA for pH values, enzyme, and their inter­
action was also conducted to identify significant factors
affecting the resulting data. The correlation was estimated
352 I. BALDERAS-LEÓN ET AL.

by the method of Pearson (P < .05) using the software
Minitab 18 (Minitab Inc., State College, PA, USA).
Results and discussion
Protein hydrolysis
Alcalase, Trypsin, and α-Chymotrypsin were used to produce
hydrolysates from LLS cotyledon globulin and glutelin protein
concentrates. The DH behaved similarly between the indepen­
dent enzymatic systems with the higher values of 78.40%,
72.71%, and 42.52% for globulins with α-Chymotrypsin,
Alcalase, and Trypsin respectively at 180 min. Simultaneously,
DH values for glutelin fraction were 82.71%, 62.75%, and 40.64%
with Alcalase, α-Chymotrypsin, and Trypsin hydrolysis at
180 min, respectively (Figure 1). These values were higher than
those reported for other non-conventional legumes like Vigna
subterranea, where maximum DH was 38%, 27.5%, and 22%
using Alcalase, Thermolysin, and Trypsin, respectively, after
24 h hydrolysis (Mune Mune et al., 2018). And, even higher
than the DH values observed for lentil hydrolysates (Lens
culinaris var. Castellana) produced by Alcalase, Savinase, and
Protamex (23%, 15%, and 11%) after 4 h of hydrolysis (Garcia-
Mora et al., 2014). The variation in DH observed in Figure 1 is
probably the result of protease specificity and protein source.
Microbial proteases such as Alcalase (an industrial alkaline pro­
tease from Bacillus licheniformis) can produce hydrolysates with
biological activities, similar to animal (gastrointestinal) Trypsin
and α-Chymotrypsin or plant-derived proteases. This is consis­
tent with previous reports, since the hydrolysates with Alcalase
exhibited the greatest DH for the underutilized legume
Macrotyloma uniflorum (45%), for Phaseolus lunatus (40%), and
Phaseolus vulgaris (50%) protein (Bhaskar et al., 2019; Torruco-
Uco et al., 2009). Furthermore, Alcalase presents broad specificity
and hydrolyzes most peptide bonds, which produce small mole­
cular weight peptides, especially those containing aromatic
amino acid residues (Phe and Tyr), and releasing peptides with
hydrophobic amino acids residues such as Trp, Leu, Ile, Val, and
Met. At the same time, Trypsin cleaves mainly C-terminal to
hydrophilic amino acid residues such as Arg and Lys.
α-Chymotrypsin hydrolyzes specifically peptide bonds formed
by C-terminal aromatic amino acids residues of Tyr, Phe, Trp, and

Figure 1. Degree of hydrolysis depending on reaction time (min) in (a) globulins and (b) glutelins of Leucaena leucocephala seed cotyledon protein concentrates
by Alcalase, Trypsin, and α-Chymotrypsin. Analysis were done in triplicates. Different letters on each line indicate significantly different at P < .05.
Figura 1. Grado de hidrólisis dependiente del tiempo de reacción (min) en (a) globulinas y (b) glutelinas de los concentrados proteicos del cotiledón de
Leucaena leucocephala con Alcalasa, Tripsina y α-quimotripsina. Los análisis de hicieron por triplicado. Diferentes letras en una misma línea indican diferencias
significativas a P < .05.
 CYTA - JOURNAL OF FOOD 353

Leu, and to a lower extent, Gln, Ser, and Thr. Several researches
reported that protein hydrolysates with a DH value around or
higher to 10% have a promising medical application usage. On
the other hand, extensive hydrolysis produced low molecular
weight peptides with hydrophobic amino acid residues asso­
ciated with bioactivities such as antioxidant or ACE inhibition),
while hydrolysates with lower DH values could be used to
improve the functional properties of flours and foods
(Clemente et al., 1999; Zhong et al., 2007). Regarding the ACE-
inhibitory bioactive peptide properties, the ACE-I prefers sub­
strates containing hydrophobic (aromatic or branched lateral
chain) amino acid residues. In contrast, hydrophobic (Ala, Leu,
Phe, Val, Pro, and Gly) and hydrophilic amino acids (Cys, Met, His,
and Ser) have been proposed to inhibit the α-Amylase activity.
Alcalase and α-Chymotrypsin are suitable for releasing
bioactive peptides, such as those with ACE-I and α-Amylase
inhibitory action. Consequently, our findings suggest that in
both LLS globulins and glutelins hydrolysates, the hydrophobic
amino acid content is comparable to that previously reported
in other protein sources when hydrolyzed with Alcalase,
α-Chymotrypsin, and Trypsin.
Antioxidant activity by DPPH
In this study, free radical scavenging activities of the
hydrolysates prepared with Alcalase, Trypsin, and
α-Chymotrypsin were determined by the DPPH• method,
used to measure the ability of antioxidant compounds to
donate electrons or hydrogen ion to free radicals to form
a stable species (Figure 2). The highest DPPH• antioxidant
activity was observed for glutelins fraction hydrolysates
using Trypsin (37.11%) and by α-Chymotrypsin
(36.43%), while the globulin hydrolysate produced by
α-Chymotrypsin showed higher activity (33.63%) than
those prepared with Alcalase and Trypsin after 180 min of
hydrolysis. Similar results were reported for Bambara
groundnut (Vigna subterranea) hydrolysates generated
with Alcalase, Trypsin, and Pepsin, being the DPPH• radical-
scavenging activity around 38% to 40% (Arise et al., 2016).
Zhang et al. (2010) reported 29% DPPH activity in alcalase
soy hydrolysates, and Valdez-Ortiz et al. (2012) reported
that Azufrado Higuera bean hydrolyzed with Alcalase
showed a 44% DPPH scavenging activity. The Alcalase

Figure 2. DPPH• antioxidant activity in (a) globulins and (b) glutelins of Leucaena leucocephala seed cotyledon protein hydrolysates by Alcalase, Trypsin, and α-
Chymotrypsin. All experiments were done in triplicates. Different letters on each line indicate significantly different at P < .05.
Figura 2. Actividad antioxidante por DPPH en (a) globulinas y (b) glutelinas en los hidrolizados proteicos de proteínas de cotiledón de Leucaena leucocephala producidos
con Alcalasa, Tripsina y α-quimotripsina. Los análisis de hicieron por triplicado. Diferentes letras en una misma línea indican diferencias significativas a P < .05.
354 I. BALDERAS-LEÓN ET AL.

hydrolysate of African yam bean seed (Sphenostylis steno­
carpa) showed around 35% of DPPH• radical-scavenging
activity (Ajibola et al., 2011). On the other hand, Pigeon
pea (Cajanus cajan) protein hydrolyzed with Pepsin-
Pancreatin presented 36.30% of DPPH• discoloration
(Olagunju et al., 2018), indicating the release and exposure
of peptides with enhanced radical scavenging activity dur­
ing enzymatic proteolysis.
Furthermore, extensive protein hydrolysis with Pepsin-
Pancreatin would likely release smaller-sized peptides that
could easily interact with free radicals. It is well known that
hydrophobic amino acids and particularly His, Pro, Met, Cys,
Tyr, Trp, and Phe may enhance bioactive peptides’ antiox­
idant activity through proton donation DPPH• radicals (Durak
et al., 2013; López-Barrios et al., 2014).
ABTS antioxidant activity
The results of ABTS˙+ scavenging activity are reported in
Figure 3. All hydrolysates from globulins and glutelins with
Alcalase, Trypsin, and α–Chymotrypsin showed high ABTS•+
scavenging activity, in the range of 60-85%, during the first
20 min of hydrolysis. These results were higher than the
53.3% of ABTS•+ scavenging activity of Pinto beans
(Phaseolus vulgaris cv. Pinto) hydrolysate after 30 min of
Protamex treatment (Ngoh & Gan, 2016). Valdez-Ortiz et al.
(2012) reported 73% and 75.3% for ABTS•+ scavenging activ­
ity of Azufrado beans (Phaseolus vulgaris) protein hydroly­
sate obtained by Pepsin and Pancreatin, respectively, after
two h of hydrolysis. Olagunju et al. (2018) reported there
was no significant difference (P < .05) between ABTS•+
scavenging activity of pancreatin (73.8%) and Pepsin-
Pancreatin (71.1%) from Pigeon pea (Cajanus cajan) hydro­
lysates (4 h of hydrolysis). The assessment of the ABTS•+
radical scavenging activity is used in both types of hydro­
philic and lipophilic compounds. A high degree of hydrolysis
and consequently high content of released amino acid
groups should increase the hydrolysates’ antioxidant capa­
city (López-Barrios et al., 2014; Torruco-Uco et al., 2009).
However, these findings suggest that the high ABTS•+ radical

Figure 3. ABTS•+ antioxidant activity in (a) globulins and (b) glutelins of Leucaena leucocephala seed cotyledon protein hydrolysates by Alcalase, Trypsin, and α-
Chymotrypsin. Experiments were done in triplicates. Different letters on each line indicate significantly different at P < .05.
Figura 3. Actividad antioxidante por ABTS en (a)globulinas y (b)glutelinas en los hidrolizados proteicos de proteínas de cotiledón de Leucaena leucocephala
producidos con Alcalasa, Tripsina y α-quimotripsina. Los análisis de hicieron por triplicado. Diferentes letras en una misma línea indican diferencias significativas
a P < .05.
 CYTA - JOURNAL OF FOOD 355

scavenging activity in the early stages of hydrolysis with
the proteases cannot be directly attributed to the DH as
the main factor but to the structure of the peptide and the
sequence of amino acid. Other factors that could modulate
the inactivation of ABTS free radical are the increase in
polarity due to the release of low-molecular-weight pep­
tides, the increase in the number of ionizable groups acting
as proton donors, and the exposure of hydrophobic side-
chain groups (Leu, Pro, His, Cys, and Met) and aromatic
amino acids (Trp, Tyr, and Phe) residues (Yi-Shen et al., 2018).
ACE inhibitory activity
The bioactive peptides with high antioxidant activity may
also be able to reduce blood pressure in cardiovascular
diseases (Martinez-Maqueda et al., 2012); thus, angiotensin-
converting enzyme (ACE) inhibitory activity of protein hydro­
lysates was also measured (Figure 4). The glutelin Alcalase
hydrolysis showed an ACE inhibitory activity of around 85%
within 60 min of reaction. It reached its highest activity
(95.44%) at 180 min, while in globulins, the Trypsin and
Alcalase hydrolysates showed an 88.47% and 86.93% of
ACE inhibitory activity, respectively. Similar results have
been observed for different protein hydrolysates, Horse
gram (Macrotyloma uniflorum) hydrolysate with a DH of
40% using Alcalase after 3 h of reaction, suggesting that
there might be an optimal DH, after which the hydrolysis
of ACE inhibitory peptides leads to the production of inac­
tive peptides or free amino acids (Bhaskar et al., 2019). Our
ACE inhibition values were significantly lower than the posi­
tive control, captopril (98.56% of ACE inhibition) but higher
than those of Lentil (Lens culinaris var. Castellana) hydrolyzed
with Alcalase (71%), Savinase (63%), Protamex (41%) and
Corolase 7089 (50%) regardless of hydrolysis time (2 h).
The ACE-inhibitory activity could be directly associated
with chain length and peptide sequence (Garcia-Mora
et al., 2014). Furthermore, Guo et al. (2018) observed that
Lupin protein hydrolyzed with Alcalase, Trypsin, and Pepsin
displayed a 56.25% within 60 min, 65% after 120 min, and
67.21% ACE inhibition at 15 min, respectively. ACE–inhibitory

Figure 4. ACE-I inhibition activity in (a) globulins and (b) glutelins of Leucaena leucocephala seed cotyledon protein hydrolysates from globulin and glutelin by
Alcalase, Trypsin, and α-Chymotrypsin. Experiments were done in triplicates. Different letters on each line indicate significantly different at P < .05.
Figura 4. Actividad inhibitoria de la enzima convertidora de angiotensina (ACE-I) en (a)globulinas y (b)glutelinas en los hidrolizados proteicos de proteínas de
cotiledón de Leucaena leucocephala producidos con Alcalasa, Tripsina y α-quimotripsina. Los análisis de hicieron por triplicado. Diferentes letras en una misma
línea indican diferencias significativas a P < .05.
356 I. BALDERAS-LEÓN ET AL.

efficiency is also associated with short peptide chains con­
taining between 3-12 amino acid residues, interacting with
the ACE-I active site’s three subsites. Aromatic and hydro­
phobic amino acids (Phe, Tyr, Pro, and Trp) have been
reported as the most favorable C-terminal residues for ACE
inhibition (Wu et al., 2006). Thus, the ACE inhibition may be
associated to the different peptide endings caused by enzy­
matic proteolysis (López-Barrios et al., 2014).
α-Amylase inhibitory activity
α-Amylase is one of the main enzymes involved in the
digestion of dietary starch, releasing oligosaccharides that
can be further broken down into glucose, which is fast
absorbed by the body. Therefore, α-Amylase activity inhibi­
tion is an effective strategy for managing blood glucose in
diabetic patients (Lammi et al., 2019). In Figure 5, results
suggested that the hydrolysis time and the kind of proteases
used were affected by cotyledon proteins’ activity. Hence,
Alcalase globulin hydrolysate (hydrolysis time from 20 min
to 180 min) displayed a higher inhibition of 74.21% at
100 min than glutelin Alcalase hydrolysate with the higher
α-Amylase inhibition activity of 58.45% at 100 min.
Furthermore, Trypsin and α-Chymotrypsin globulins hydro­
lysates reach the highest α-Amylase inhibitory activity up to
48.42% and 55.43% within 80 and 100 min, respectively; but
displayed a slight decline at 120 min. However, after 180 min
of the enzymatic reaction, lower α-Amylase inhibition activ­
ity and significant differences were found, whereas Alcalase
glutelins treatment exhibited a maximum value at 100 min
(91.51%). The α-Chymotrypsin and Trypsin glutelin hydroly­
sis gradually increased α-Amylase inhibitory activity of
hydrolysates. In contrast, hydrolysis of more than 60 and
100 min resulted in a rapid reduction of α-Amylase inhibition
activity.
The high α-Amylase inhibitory activity for globulin
α-Chymotrypsin and glutelin Alcalase hydrolysates could be
due to the release of a smaller and narrower-size range of

Figure 5. α-Amylase inhibition activity in (a) globulins and (b) glutelins of Leucaena leucocephala seed cotyledon protein hydrolysates from globulin and glutelin
by Alcalase, Trypsin, and α-Chymotrypsin. Experiments were done in triplicates. Different letters on each line indicate significantly different at P < .05.
Figura 5. Actividad inhibitoria de la α-amilasa en (a)globulinas y (b)glutelinas en los hidrolizados proteicos de proteínas de cotiledón de Leucaena leucocephala
producidos con Alcalasa, Tripsina y α-quimotripsina. Los análisis de hicieron por triplicado. Diferentes letras en una misma línea indican diferencias significativas
a P < .05.
 CYTA - JOURNAL OF FOOD 357

peptides generated within 20 min to 100 min of reaction. It of using LLS proteins for food purposes, with minimal mimo­
was proposed that these peptides were bound at the sub­ sine toxicity. The mimosine concentration decreases up to
strate-binding site of α-Amylase, affecting the interaction to 64.92% in the next stepwise, the end of the protein hydrolysis
the carbohydrate by steric hindrance. Hydrophobic interac­ reaction, especially in the glutelin fraction treated with Trypsin
tions may also contribute to conformational changes on the compared to the initial values. Thus, protein isolation and
substrate-binding region of α-Amylase, inhibiting the hydro­ subsequent enzymatic hydrolysis could be considered as
lysis process (Awosika & Aluko, 2019). These results are con­ a way to utilize LLS cotyledon proteins with minimal interfer­
sistent with those reported by Oseguera-Toledo et al. (2015), ence from mimosine, avoiding its toxicity complications.
where the non-hydrolyzed protein had around 30% of
α-Amylase inhibition. After 120 min of hydrolysis, all hydro­
lysates showed the highest inhibition of α-Amylase, approxi­ Correlation coefficients
mately 67%. These promising results indicate that LLS
According to Table 2, there was a significant correlation
cotyledon could be a protein source to produce bioactive
between DH and all bioactivities of LLS cotyledon hydrolysates.
peptides with potential anti-diabetic properties.
The two-way ANOVA also indicated highly significant differ­
ences (P < .0001) between the reaction time and the kind of
Mimosine content enzyme used. Thus, this might be the main contributor to the
antioxidant, anti-α-Amylase, and anti-ACE activities of LLS coty­
Table 1 shows the mimosine concentration in Leucaena leuco­
ledon hydrolysates. Antioxidant assays, including ABTS•+, were
cephala seeds (LLS) cotyledon, protein concentrates, hydroly­
especially strongly correlated with the anti-ACE and relatively
sates, and the percentage of mimosine reduction (%). Most
strongly correlated to anti-α-Amylase activity assays at P < .05.
mimosine was extracted in the first step during the protein
Meanwhile, the DPPH• assay was not significantly associated
concentrates preparation, either with NaCl 5% or NaOH 1 N.
with the α-Amylase inhibitory assay.
After acid precipitation, mimosine was reduced by 87.66%.
These results agree with previous reports (Ekpenyong, 1986;
Rodrigues-Corrêa et al., 2019; Sethi & Kulkarni, 1993). The LLS Conclusions
mimosine concentration varies widely among the various cul­
tivars of the species. Mimosine accounts for approximately 60% This study observed that protein hydrolysates derived from
of the total free amino acids in LLS (Sethi & Kulkarni, 1995). The Leucaena leucocephala seed cotyledon possess antioxidant
reduction in mimosine content could be related to its small size properties against ABTS•+ and DPPH• radicals. They pre­
and high water solubility. Several reports claim that the pre­ sented the potential to inhibit enzymes related to chronic
paration of seed-protein concentrates yields proteins relatively diseases as hypertension (ACE-I) and type-2 diabetes (rele­
free of some inherent toxic constitutes (Bhat & Karim, 2009). vant to the effect on α-Amylase). These functions could
LLS protein isolates prepared by isoelectric precipitation potentially find promising applications as nutraceutical
yielded relatively low mimosine levels (Sethi & Kulkarni, 1993). ingredients to be used in functional foods to prevent and/
Thus, protein isolate preparation could be considered a means or treat oxidative stress which constitutes relevant patho­
physiology in many chronic diseases and provides added
value to this crop. Therefore, protein isolation by isoelectric
Table 1. Mimosine content and percentage of mimosine reduction (%) in
extracts from cotyledon flour, protein (globulin and glutelin) concentrates and
precipitation could be considered a means of utilizing
hydrolysates from Leucaena leucocephala. L. leucocephala seed protein, with minimal interference
Tabla 1. Contenido y porcentaje de reducción (%) de mimosina en los
from mimosine. Further research is required to isolate and
extractos de harina de cotiledón, concentrados proteicos (globulinas characterize bioactive peptides with potent antioxidant, ACE,
y glutelinas) e hidrolizados de Leucaena leucocephala. and α-Amylase inhibitory activity from Leucaena protein
Mimosine hydrolysates.
content Percentage of mimosine
Sample (mg/100 g) a reduction (%)
Saline soluble 150.81 ± 6.24a - Table 2. Correlation table of bioactivity of the produced hydrolysates.
extractb Tabla 2. Tabla de correlación de bioactividad de los hidrolizados proteicos.
Alkali soluble extractc 146.82 ± 5.88a -
Globulin concentrate 18.61 ± 0.08b 87.66 DHa DPPHb ABTSc ACE-Id
Glutelin concentrate 16.16 ± 1.34c 88.98 DPPH 0.588730
Globulin hydrolysates <0.0001
Before hydrolysis 13.95 ± 1.58d - ABTS 0.573529 0.270732
Alcalase 10.71 ± 0.29e 42.41 <0.0001 <0.0001
Trypsin 10.92 ± 0.37e 41.36 ACE-I 0.658026 0.318252 0.879678
α-Chymotrypsin 10.88 ± 0.37e 41.48 <0.0001 <0.0001 <0.0001
Glutelin hydrolysates e
α-Amylase-I 0.382370 −0.089332 0.616322 0.628886
Before hydrolysis 12.65 ± 0.47de - <0.0001 0.0906 <0.0001 <0.0001
Alcalase 5.97 ± 0.92f 63.03
Trypsin 5.67 ± 0.11f 64.92 Cell Contents: Pearson correlation.
α-Chymotrypsin 7.67 ± 0.39f 52.52 P-Value.
a
a
Means in the same column, followed by different letters, are significantly DH: the degree of hydrolysis, bDPPH: DPPH• scavenging activity, cABTS: ABTS•
+
different (Tukey, P < .05). Experiments were done in triplicates. scavenging activity, dACE-I: angiotensin converting enzyme inhibitory
b
5.0% NaCl cotyledon extract. activity, eAmylase-I: α-Amylase inhibitory activity.
c
1.0 N NaOH pH 8 cotyledon extract solution. Contenido en las celdas: Valor de la correlación de Pearson.
a
Los promedios en una misma columna con letras diferentes son significati­ Valor de p.
a
vamente diferentes. Todos los experimentos se hicieron por triplicado. DH: grado de hidrólisis, bDPPH: capacidad para atrapar al radical DPPH,
c
b
Concentrado proteico extraído con NaCl al 5.0%. ABTS: actividad antioxidante por ABTS, dACE-I: actividad inhibitoria de la
c enzima convertidora de angiotensina, eAmylase-I: actividad inhibitoria de la
Concentrado proteico extraído con NaOH 1.0 N NaOH a pH 8. α-Amilasa.
358 I. BALDERAS-LEÓN ET AL.
Acknowledgments
The first author acknowledges Consejo Nacional de Ciencia y Tecnología
(CONACYT) for scholarship No. 305253.
Disclosure statement
No potential conflict of interest was reported by the author(s).
ORCID
Iván Balderas-León http://orcid.org/0000-0002-4903-1743
Diana Baigts-Allende http://orcid.org/0000-0001-6728-5141
Anaberta Cardador-Martínez http://orcid.org/0000-0003-2942-1186
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