Chemical Papers

https://doi.org/10.1007/s11696-024-03492-5

ORIGINAL PAPER

Sustainable approach from underutilized Leucaena leucocephala

biomass by polyphenols composition and protein functional
properties assessment
Iván Balderas‑León1 · Anaberta Cardador‑Martínez2 · Diana Karina Baigts‑Allende3 ·
Carlos Arnulfo Velázquez‑Carriles1 · Jorge Manuel Silva‑Jara1

Received: 9 February 2024 / Accepted: 28 April 2024

© The Author(s), under exclusive licence to the Institute of Chemistry, Slovak Academy of Sciences 2024

Abstract
The seeds of Leucaena leucocephala (LL), a legume tree native to Mexico, are traditionally harvested and utilized as food
ingredients in regional dishes. In this study, the usefulness of LL biomass was investigated and processed in flours using the
wholegrain seed (WS) and pods for polyphenols identification, whereas the cotyledon (CT) for protein concentrates functional
characterization was used. The WS flour exhibited carbohydrates as the main macronutrient, followed by proteins (30.59%).
Water-extracted polyphenols from pods flour (544.29 mg/L) were higher compared to the WS flour (165.85 mg/L). The
predominant polyphenols in all samples were flavonols, specifically catechins. Although, in comparison to the mixtures of
methanol–water (315.14 mg/L) and ethanol–water (471.22 mg/L), pods had about threefold polyphenols content than WS
flours extracts. Protein fractionation in CT was higher than in WS flour, with globulins being the most abundant fraction
in both materials (44.73 and 55.2%, respectively). Structural analyzes indicated a higher presence of structural elements
in globulins, evident in FTIR spectra and SDS-PAGE analysis. Globulins displayed bands around 50, 37, and 22 kDa,
while glutelins exhibited a unique band at 50 kDa. Globulins also demonstrated a higher essential amino acid content. The
functional properties of globulins and glutelins were pH-dependent. Globulins solubility (72.53%), emulsifying (35.13%),
foaming (138.66%), water holding (3.17 g/g), and oil absorption (1.20 g/g) capacities were higher compared to glutelins
(62.42%, 31.69%, 66.70%, 2.86 g/g, and 0.52 g/g, respectively). These findings suggest that LL biomass holds potential as
a sustainable source of natural ingredients for food development.

Keywords Plant protein · Legume · Protein solubility · Chemical composition

Introduction
In recent years, the increasing response to worldwide food
demand and environmental awareness has triggered an active
search for more sustainable protein sources to satisfy the
* Jorge Manuel Silva‑Jara
jorge.silva@academicos.udg.mx
1
Departamento de Farmacobiología, Centro Universitario
de Ciencias Exactas E Ingenierías (CUCEI), Universidad
de Guadalajara, Blvd. Marcelino García Barragán #1421,
CP 44430 Guadalajara, Jalisco, México
2 nutritional quality and improve sensorial properties (Chen
Departamento Regional de Bioingenierías, Tecnológico de
Monterrey, Querétaro, México
3 proteins from less conventional legume sources, such as
Faculty of Agrobiology, Food and Natural Resources, Czech
University of Life Sciences Prague, Kamýcká 129, 165 00,
Prague‑Suchdol, Czech Republic
food industry for human nutrition (Bhat and Karim 2009).
The global market of protein as an ingredient was valued at
USD 38 billion in 2019, and is expected to grow 9.1% from
2020 to 2027 (Ismail et al. 2020). The transition of non-ani-
mal-derived proteins to plant-based ones has been promoted,
especially underutilized sources such as native crops.
Most studied plant-based sources are legumes due to
their high protein content, availability, and low cost. They
have been used as ingredients for food product develop-
ment, mainly in developing countries (Piornos et al. 2015).
Pulse proteins have been used for the production of protein
concentrates or isolates in processed food to both enhance
et al. 2017; Shevkani et al. 2015). Even though some
Mucuna pruriens and Dolichos lablab, have been explored
(Adebowale and Lawal 2003; Habib et al. 2017), there is

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little information about other autochthonous species like
Leucaena leucocephala (LL).
The LL, also known as “guaje,” is a native legume
from Mexico that belongs to the Fabaceae family. In some
regional zones, in the southeast of Mexico, it is considered
a nutritious food, which is consumed either fresh or as an
ingredient in the preparation of traditional dishes such as
sauces and stews; however, it is considered an underu-
tilized crop. Despite being consumed locally in certain
regions, LL remains largely overlooked as a viable protein
source. The seeds, particularly the cotyledons (CT), boast
a high protein content and have shown promise in protein
isolate production, offering favorable amino acid profiles
suitable for human nutrition. Additionally, LL proteins
exhibit techno-functional properties, making them attrac-
tive as additives in food formulations seeking alternatives
to animal protein (Rodrigues-Corrêa et al. 2019). This
species is also farmed for multipurpose, including green
manure, livestock fodder, and soil conservation in some
tropical areas like South America, Western Africa, and
India, where the crop is harvested (Verma et al. 2018).
The seeds are the edible part of this crop, which con-
sists of a CT (high protein content, 25–32%) covered
by an endosperm (polysaccharides, 20–25%) (Sethi and
Kulkarni 1993, 1995). The LL-CT protein isolates have
shown up to 76% purity and contain some essential amino
acids such as histidine, isoleucine, leucine, and phenylala-
nine in recommended amounts for human nutrition (Sethi
and Kulkarni 1994b). Besides their nutritional quality,
proteins are recognized as techno-functional ingredients
(thickeners, emulsifiers, stabilizers, among others) in food
product development. Thus, this protein transition is also
focused on finding sustainable additives for animal protein
replacement for different organoleptic features and even
eating habits (e.g., religion, veganism) (Ahnen et al. 2019;
Klongklaew et al. 2023).
Other nutritional constituents in plants are phytochemi-
cals, mainly the phenolic compounds, which are the most
abundant group of secondary metabolites found particu-
larly in legume sources. Their antioxidant capacity has
been related to reducing the oxidative damage associated
with some diseases, such as cancer and cardiovascular
problems (Singh et al. 2017). These biocompouds are
present in the hulls of the LL, but they are poorly studied
as a source of nutraceutical ingredients from wasted crop
biomass (Benjakul et al. 2014).
Herein, we studied the effective use of LL crops for
obtaining nutritional compounds such as protein concen-
trates and polyphenols as potential novel additives for food
applications. LL was used to obtain different flours which
were then characterized and their techno-functional prop-
erties evaluated.
Chemical Papers
Experimental
Materials
The LL pods were purchased in a local market in Oax-
aca, Oaxaca, Mexico. The sunflower oil used for some
protein functional properties was purchased from a local
brand (Campo Vivo™, Mexico). All chemicals used were
of analytical grade: methanol, ethanol, acetonitrile, gla-
cial acetic acid, formic acid, hydrochloric acid, sodium
hydroxide, sodium chloride, bovine serum albumin (BSA),
borate buffer, sodium phosphate monobasic monohydrate
­(NaH2PO4·H2O), nitrogen gas, Laemmli buffer, polyacryla-
mide gels, Coomassie Blue acetic, polyphenols standards
(gallic acid, vanillic acid, caffeic acid, chlorogenic acid,
ρ-coumaric acid, and epicatechin), amino acid standards,
o-phthalaldehyde (OPA) and 9-fluorenylmethyl chloro-
formate (FMOC), were obtained from Sigma-Aldrich (St.
Louis, MO). HPLC-grade water was generated by a Milli-Q
system (Millipore, Bedford, MA, USA). The LL samples
were immediately stored at − 20 °C in airtight dark contain-
ers prior to extractions.
Proximate analysis
The proximal composition of the LL seeds was determined
by the standard methods of AOAC (2016). Moisture con-
tent (930.15), ash (923.03), protein (976.05) (N × 6.25), total
dietary fiber (958.29), and fat (920.39). Carbohydrates were
determined by weight difference.
Preparation of material
The seeds and pods from LL were used for the preparation
of three different flours: wholegrain seeds (WS), seeds pods,
hull seed, and CT. For WS and CT, the seeds were soaked in
water for 2 h; then, the hulls were manually removed from
the swollen CT using a scalpel. WS and CT, and seeds pods
were air-dried at 60 ºC for 48 h (Binder™ ED 23, Germany)
and ground in an electric miller (Krups™ GX4100, Ger-
many) to obtain the flours. Afterward, the flours were sieved
through a 625 μm mesh and stored at 4 ºC until use.
Quantification of polyphenols
WS and seed pods materials were used for polyphenols
extraction using three solvents: (a) water (60 °C), (b) metha-
nol–water (80:20), and (c) ethanol–water (80:20) (b and c at
room temperature, 25 °C). The extractions were carried out
for 30 min using a 1:10 solute:solvent ratio. Afterward, the
extracts were filtered using both Whatman filter paper No.
Chemical Papers
40 and nylon syringe filter (0.2 μm). An LC–MS/MS equip-
ment (Waters, Milford, MA, USA) with an Acquity UPLC
BEH C18 column (2.1 × 100 mm, 1.7 μm) was used for the
identification and quantification of polyphenols. An Acquity
UPLC BEH C18 (2.1 × 100 mm, 1.7 μm) was used for com-
pound separation. Mobile phases were 0.1% (v/v) formic
acid in water (A) and 0.1% (v/v) formic acid in methanol
(B). The column was maintained at 40 °C. A flow rate of
0.45 mL/min and an injection volume of 5 μL were used.
Total run time of 14.5 min with 99% A at 0.5 min, 20% A at
13.5 min, and 99% A at 14 min was used. Quattro Premier
XE triple quadrupole mass spectrometer fitted with electro-
spray ionization (ESI) was used in a negative mode for gallic
acid, vanillic acid, caffeic acid, chlorogenic acid, ρ-coumaric
acid, and epicatechin. Mass spectrometer optimization was
conducted using 50 mg/L stock standard solutions to select
the ionization mode and precursor and product ions. The
calibration curves (3–50 µg/mL) of the polyphenol stand-
ards mentioned were used for quantification. Polyphenols
were identified by their retention time, MS-fragmentation
patterns, and comparison to standards.
Leucaena leucocephala seeds protein fractionation
The LL seeds protein fractions from WS and CT were
obtained according to the sequential fractionation Osborne
method as described by Teka et al. (2020) Solutions of
flour and distilled water (1:10 w/v) were stirred for two h at
room temperature, followed by centrifugation (Heraeus™
Multifuge™ X1R) at 5000 × gfor 15 min at 4 °C. The first
supernatant (albumin fraction) was collected and stored at
4 °C. The remnant pellets for the next sequential extrac-
tions were solubilized with NaCl (5%), NaOH (pH 8), and
ethanol (70%) to obtain globulins, glutelins, and prolamins
fractions, respectively. Mixtures were stirred and centrifuged
as described before. The protein extractions were performed
in triplicate. The content of protein in the protein fraction
extracts was determined by the Bradford microplate assay,
using bovine serum albumin (BSA) as standard for the cali-
bration curve (Bradford 1976). Based on the protein frac-
tion yields, CT flour was the selected material for further
analysis.
Preparation of protein concentrates (globulins
and glutelins)
Globulins and glutelins proteins from extracts previously
described were precipitated at their isoelectric point (IEP)
using a solution of HCl (0.1 N, pH 6) and recovered by cen-
trifugation at 5000 × g for 15 min at 4 ºC (Rodríguez-Ambriz
et al. 2005). The protein extraction from the residual pellet
was carried out twice. The precipitates from both extrac-
tions for globulins and glutelins fractions were washed,
neutralized, and freeze-dried (Labconco™ Triad 740040,
USA).
Amino acid analysis
Amino acids (AA) were determined by HPLC according
to the Agilent method (Woodward et al. 2007). The protein
concentrates samples (0.2 g) were subjected to acid hydroly-
sis with 25 mL of 6 N HCl under a nitrogen atmosphere for
24 h at 110 °C. The resulting hydrolysate solutions were
adjusted to pH 7 and washed with distilled water, then fil-
tered through a 0.22 µm syringe filter. Before injection, sam-
ples were automatically derivatized online using OPA or
with FMOC. The equipment was injected by programming
the autosampler with volumes of 2.5 μL of borate buffer
mixed with 0.5 μL of sample, 0.5 μL of OPA, 0.5 μL of
FMOC reagent, and 32 μL of HPLC-grade water. The analy-
sis was performed on an Agilent 1100 system (Agilent Tech-
nologies, Waldbronn, Germany), composed of a µ-degasser
(G1379B), 1260 binary pump (G1312B), 1260 standard
autosampler (G1329B), 1260 thermostated column compart-
ment (G1316A), 1260 diode array and multiple wavelength
detector (G1315C), and a Zorbax Eclipse-AAA column
(150 mm × 4.6 mm, i.d., particle size 5 μm) (Agilent Tech-
nologies, Santa Clara, CA). The column temperature was
set at 40 °C, with detection at λ1 = 338 nm (for OPA-amino
acids) and λ2 = 262 nm (for FMOC-amino acids). The mobile
phase comprised a 40 mM ­NaH2PO4·H2O pH 7.8 solution
(mobile phase A) and a mixture of water, methanol, and
acetonitrile (10:45:45 v/v/v; phase B). Gradient samples elu-
tion at a flow rate of 2 mL/min were: 0 min, 0% B; 1.9 min,
0% B; 18.1 min, 57% B; 18.6 min, 100% B; 22.3 min, 100%
B; 23.2 min, 0% B; and 26 min, 0% B. Amino acid stand-
ards were used to identify each amino acid by their retention
times (Sigma-Aldrich, St. Louis, MO).
SDS‑PAGE analysis
The determination of the electrophoretic profile of the pro-
tein concentrates was performed according to the method
described by Laemmli (1970). Protein samples (5 mg) were
dissolved in 1 mL of Laemmli buffer (0.5 M Tris–HCl (pH
6.8), 10% SDS, 10% glycerol, 5% b-mercaptoethanol, and
0.1% bromophenol blue) and heated at 90 °C for 5 min.
Afterward, aliquots of 5 μL were poured into polyacryla-
mide gels (4% stacking; 8–16% separating), and run at con-
stant voltage (60 V). The gels were stained with Coomassie
Blue for 15 min and destained with methanol:water:acetic
acid (3:6:1 v/v/v) solution. The destained gels were pho-
tographed using an iBright™ CL1000 Imaging System
(Thermo Fisher Scientific, Massachusetts, United States).
A molecular weight standard (6.5–66 kDa, Sigma-Aldrich,
St. Louis, MO, USA) was used as a marker.

Fourier transform infrared (FTIR)
Infrared spectra of globulins and glutelins were recorded using
Fourier transform infrared (FTIR) spectrometer (Agilent Cary
630 spectrometer, Agilent Technologies, USA) equipped with
Attenuated Total Reflectance (ATR) cell (PIKE Technology
Inc., USA). For spectra analysis, protein concentrates samples
were placed on the crystal cell, and the cell was gripped into
the mount of the FTIR spectrometer. Spectra were measured
in the range of 500–4000 ­cm−1 using 32 scans at a resolution
of 4 ­cm−1 against a background spectrum recorded from the
clean empty cell at room temperature.
Techno‑functional properties of Leucaena
leucocephala protein fraction concentrate
Protein solubility
Protein dispersions (1% w/v) were prepared at different pH
values (from 2 to 10) using 0.1 N HCl or NaOH solutions. The
dispersions were stirred for 2 h at room temperature and centri-
fuged (5000 × g for 15 min at 4ºC). The supernatant was used
for protein quantification by Bradford (1976) microplate assay
method (xMark™ Microplate Spectrophotometer). Control
samples were prepared dissolving the protein in 0.5 N NaOH
(pH 12). All samples were measured in triplicate, percent-
age of soluble protein was calculated using Eq. 1 (Rodríguez-
Ambriz et al. 2005):
Protein in supernatant
Solubility (% )= × 100
Total protein content in control
Emulsifying capacity (EC) and emulsion thermal stability
(ES)
Protein emulsions were prepared by homogenization of
10 mL of protein solutions (1% w/v) and 5 mL of sunflower
oil using an Ultra-Turrax (T18, IKA Instruments, Germany) at
12,500 rpm for 5 min at room temperature. Next, the emulsify-
ing capacity (EC) after the application of gravitational forces
(1300 × g for 5 min at 4ºC) was measured using Eq. 2 (Karaca
et al. 2011). A similar procedure was followed to determine
the emulsifying stability (ES), incubating the samples at 80 °C
in a water bath for 30 min before centrifugation. The ES was
calculated with Eq. 3.
Height of emulsified layer in the tube
Emulsifying capacity (% ) =
Height of the total content in the tube
Height of emulsified layer after heating
Emulsifying stability (% ) =
Height of the emulsion before heating
Chemical Papers
Foaming capacity (FC) and foam stability (FS)
The foaming capacity (FC) and foaming stability (FS) of
proteins were determined, according to Piornos et al. (2015).
Protein solutions at 1% w/v were blended in a homogenizer
(Ultra-Turrax T18, IKA Instruments, Germany) at 12,500 rpm
for 1 min at room temperature. After homogenization, the dis-
persion was poured in a test tube and the height of the foam
present above the surface of the liquid was measured. For
FS, the sample was allowed to stand up and monitored for
120 min. FC and FS were calculated with Eq. 4.
V2 − V1
Foaming capacity (% ) = × 100 (4)
V1
where V2 is the volume of protein solution after homogeni-
zation, and V1 is the original volume of protein solution.
Additionally, the effect of pH on foaming properties (from
2 to 10) was also evaluated. Experiments were performed
in triplicate.
Water holding capacity (WHC) and Oil absorption
capacity (OAC)
To assess both the water holding capacity (WHC) and oil
absorption capacity (OAC), the protein concentrates (1 g) were
dispersed in 10 mL of either distilled water or sunflower oil
within a pre-weighed 15 mL centrifuge tube. The dispersions
were thoroughly vortexed (VV3, VWR, Lutterworth, UK), and
the samples were left to stand at room temperature for 30 min
before being centrifuged (5000 × g, 15 min, 4 °C). Following
(1) centrifugation, the supernatants were discarded, and the cen-
trifuge tubes containing the sediments were weighed (Mune
Mune and Sogi 2015). WHC was reported as grams of water
absorbed per gram of protein concentrate. OAC was reported
as grams of water or oil absorbed per gram of protein concen-
trate, determined using Eqs. 5 and 6, respectively.
M 2 − M1
WHC (g hold water /g protein)= (5)
W
M2 − M1
OAC (g absorbed oil /g protein) = (6)
W
where W was the weight of the dry protein concentrate sam-
ple (g), M1 was the weight of the tube plus the dry sample
(g), and M2 was the weight of the tube plus the sediment (g)
after the decantation of water or oil.
× 100 (2)
Statistical analysis
× 100
The extractions and experiment replicates were conducted
(3) in triplicate, and the values were expressed as mean ± stand-
ard deviation. Data analysis involved one-way ANOVA, and
Chemical Papers
mean comparison was executed using Tukey’s test (statisti-
cal significance considered at P < 0.05). Minitab 18 (Minitab
Inc., State College, PA, USA) was utilized for statistical
analysis, and GraphPad Prism 9 was employed for creating
the plots.
Results and discussion
Leucaena leucocephala seeds proximate analysis
The proximal analysis of LL seeds is shown in Table 1. It
was observed that after the carbohydrates, the proteins were
the main constituent (~ 30%). The protein content agreed
with values reported for the same species harvested in dif-
ferent regions of Africa, such as Western Nigeria, ~ 32%
(Adeneye 1979), Nigeria, ~ 25–30% (Ekpenyong 1986),
and Sudan, ~ 31% (Ahmed and Abdelati 2009). Interest-
ingly, the content of protein was higher in comparison to
other non-conventional legume crops like Lablab purpureus,
23% (Hossain et al. 2016); Vigna unguiculata, 26% (Rivas-
Vega et al. 2006); Canavalia ensiformis, 27% (Rajaram and
Janardhanan 1992); Prosopis juliflora, 28% (Ortega-Nieb-
las et al. 1996) and Phaseolus acutifolius, 23% (Porch et al.
2016). This high protein concentration indicates that LL
seeds might be used as a considerable protein supplement
in a variety of diets, especially in areas where protein-rich
food sources are limited. Regarding the fat and carbohy-
drates, the results (4.04 and 55.38%, respectively) align
with values previously reported for LL seeds, albeit slightly
lower than some reported ranges (5–10% fat and 35–45%
carbohydrates) (Alemán-Ramirez et al. 2022; Garcia et al.
1996; Sethi and Kulkarni 1994a, 1995). Climate, harvest
conditions, soil characteristics, and genetic traits all have
an impact on composition, which varies across regions.
Understanding these variations is crucial for optimizing LL
cultivation and utilization, especially in agricultural and
nutritional contexts. Additionally, further research into the
nutritional composition of LL seeds under different growth
stages and conditions could provide valuable insights into
enhancing their nutritional value and utilization potential
(Porch et al. 2016; Rui et al. 2011).
Table 1  Proximate composition of whole Leucaena leucocephala
seed flour (dry weight basis)
Component Composition (%)
Protein (N × 6.25) 30.59 ± 0.79
Fiber 8.33 ± 0.29
Ash 1.66 ± 0.11
Fat 4.04 ± 0.33
Carbohydrates (by difference) 55.38 ± 0.10
Identification and quantification of polyphenols
in Leucaena leucocephala flours
Seven polyphenolic compounds were identified and quanti-
fied from WS and seed pod flours, including two hydroxy-
benzoic acids (HBA) (gallic and vanillic acid), three hydrox-
ycinnamic acids (HCA) (caffeic acid, 3-caffeoylquinic acid,
and p-coumaric acid), and two flavanols (FLA) (epicatechin
and catechin) (Table 2). For both WS and seed pods, the
highest extraction of polyphenols resulted using water as sol-
vent (~ 165 and 544 mg/L, respectively) followed by the mix-
tures ethanol:water (160 and 471 mg/L) and methanol:water
(100 and 315 mg/L). This behavior has already been
reported, where the extraction of polyphenols is driven by
their solubility and structural composition (polarity), find-
ing them more soluble in water than in organic solvents.
The highest water-extracted FLA compounds were around
68–81 and 74–88% for WS and seed pods, respectively. For
HBA, both samples showed similar values (~ 6–16%), and in
the case of HCA, WS had slightly higher amounts (11–17%)
than seed pods (7–10%). In general, the seed pods showed
a total content of polyphenols around three times more than
WS. Total polyphenol content in LL seeds has been reported
only using indirect methods (~ 37 mg GAE/g) (Chowtivan-
nakul et al. 2016), however, so far, there are no studies on
the identification and quantification of polyphenols using
LC–MS/MS chromatographic techniques in LL seeds and
seeds pods. In comparison to other legumes, such as black
and ruviotto beans (Phaseolus vulgaris) with concentrations
of 459 and 189 mg/Kg, respectively, black lentils (Vigna
mungo) exhibit a polyphenol content of 137 mg/Kg, and
gold lentils (Lens esculenta) of 132 mg/Kg (Caprioli et al.
2018). These findings indicate a concordance in terms of
their polyphenol content within the water WS and seed pod
extracts of LL. Comparing the polyphenol concentration
of LL to other legumes provides insight into its nutritional
profile among widely consumed meals. While LL polyphe-
nol levels are comparable to those of other legumes, its dis-
tinct polyphenolic composition may provide unique health
benefits.
The identification and quantification of polyphenolic
compounds in LL flours provided insight on their potential
health benefits and nutritional significance. Polyphenols are
recognized for their antioxidant properties and have been
associated with various health-promoting effects, includ-
ing anti-inflammatory and anti-cancer activities (Bhat and
Karim 2009). The presence of HBA, HCA, and FLA in LL
flours indicates their rich polyphenolic profile, which could
contribute to its nutritional value. The choice of solvent
for polyphenol extraction, with water yielding the highest
extraction efficiency, underscores the importance of solvent
selection in maximizing polyphenol recovery. Variations in
the concentration of bioactive substances across different
 Chemical Papers

Table 2  Polyphenols identified in the extracts from seeds and seed pods of Leucaena leucocephala
Compound [M– Fragments m/z Concentration (mg/L)
H] − m/z (MS2) (intensity)
(MS)
SW SM SE PW PM PE

Hydroxybenzoic acids (HBA)

Gallic ­acida 169 125 (100) 27.02 ± 0.81c 15.37 ± 0.62 13.55 ± 0.21 76.99 ± 3.44 42.74 ± 0.54 20.84 ± 0.62
Vanillic ­acida 167 152 (100) ndb nd nd 5.77 ± 0.18 5.51 ± 0.13 5.09 ± 0.16
Hydroxycinnamic acids (HCA)
Caffeic ­acida 179 135 (100) 14.95 ± 0.64 7.40 ± 0.28 6.98 ± 0.18 10.26 ± 0.46 5.20 ± 0.14 7.50 ± 0.28
3-Caffeoylquinic 353 179 (100), 135 nd nd nd 5.84 ± 0.12 12.45 ± 0.21 5.12 ± 0.17
­acida (64), 125 (52)
p-Coumaric ­acida 163 119 (100) 10.01 ± 0.20 9.78 ± 1.72 10.29 ± 0.42 25.22 ± 0.33 14.97 ± 0.66 19.07 ± 0.70
Flavanols (FLA)
Epicatechina 291 139 (100) 4.87 ± 0.10 15.44 ± 0.65 15.40 ± 0.57 4.83 ± 0.18 5.82 ± 0.11 5.90 ± 0.28
Catechin 291 139 (100) 109.01 ± 2.17 52.70 ± 2.24 113.85 ± 2.62 415.41 ± 6.82 228.46 ± 5.83 407.71 ± 4.04
Total 165.85 ± 3.72 100.68 ± 4.94 160.05 ± 4.00 544.29 ± 10.27 315.14 ± 7.22 471.22 ± 5.34
a
Identified and quantified with authentic standards. bnd = not detected. cResults expressed in mg/L of extract. Data are presented as mean ± SD
(n = 3)
SW Whole seed flour (water), SM Whole seed flour (80:20 methanol:water), SE = Whole seed flour (80:20 ethanol:water). PW Pod flour (water),
PM Pod flour (80:20 methanol:water), PE Pod flour (80:20 ethanol:water)

portions of the LL plant, such as higher polyphenol levels 60 a

in seed pods than in seeds, indicate prospects for targeted 50 b

extraction and use of certain plant sections to improve their Albumins
40 c
nutritional value. Globulins
Fraction %

a Glutelins
30
Prolamins
Structural characterization of Leucaena 20 b
b a c
leucocephala storage proteins c
10
a a a
Protein fractioning 0
Ctrl DF CT Ctrl DF CT Ctrl DF CT Ctrl DF CT
Material
The protein content found in CT was higher than WS flour
(Control) and defatted flour (DF); globulins were the most Ctrl: whole seed flour, DF: seed defatted flour and CT: cotyledon flour

abundant fraction in the three flours (55.2, 44.73, and ~ 35%,

respectively), as shown in Fig. 1. Although specific data
on LL seed protein fractions are scarce in the literature,
the prevalence of globulins in both CT and WS flours line
up with typical legume protein profiles, where these pro-
teins generally contribute over 70% of total protein content.
Globulins are recognized for their solubility in water-based
conditions and serve important roles in a variety of bio-
logical processes. Their abundance implies that they may
have functional functions in LL seeds, such as nutrient
storage, seed germination, and plant defense mechanisms
(Makeri et al. 2017; Meng and Ma 2002). Comparable frac-
tions have been reported in other legumes such as Sesbania
pachycarpa, Milletia thonningii, Mucuna utilis, and Mucuna
cochinchinensis, where similar globulins fractions (38.63,
54.23, 63.75, and 58.30%, respectively) have been shown.
Nevertheless, in Adenanthera pavonina, Prosopis africana,
and Lonchocarpus sericeus, the major fraction was glutelins
Fig. 1  Leucaena leucocephala protein fractions for whole seed flour
(Control), seed defatted flour (DF), and cotyledon (CT). Different
letters on the column top indicate the significant difference (Tukey's
HSD test, P < 0.05), among different materials (at a given protein
kind). Data points represent means (n = 3) ± SD
(44.17, 48.53 and 52.55%, respectively) (Ezeagu and Gowda
2006). In contrast, prolamins constitute the lowest fraction
in both CT and WS flours (2.2 and 2.18%, respectively),
consistent with their minor role in legume protein composi-
tion. These low content of prolamins corresponded to Vinga
angularis (~ 2–5%) (Durak et al. 2013) and Psophocarpus
tetragonolobus (~ 4%) (Yea et al. 2014). Comparing differ-
ent legume species reveals differences in protein fraction
patterns, indicating the plant family diversity. The pres-
ence of similar globulin fractions across different legumes
underlines the conserved nature of globulins among them.
Chemical Papers

Based on total protein content and protein fractions yield, Table 3  Amino acid composition of Leucaena leucocephala cotyle-
globulins and glutelin from CT samples were selected for don protein fractions
further analysis. This selection is based on their abundance Amino acid Concentration (g/100 g protein)
and possible functional properties, allowing opportunities
Globulins Glutelins FAO/WHO,
for studying the nutritional importance and application of 2007
LL storage proteins. Adult
The amino acid composition of protein fractions from recommen-
dations
LL seeds cotyledon offers valuable information regarding
their nutritional quality and potential applications in food *His 1.27 ± 0.01 3.00 ± 0.01 1.6
industries. Table 3 illustrates the amino acid profiles of *Ile 7.35 ± 0.21 8.81 ± 0.04 3.0
globulins and glutelins from CT protein concentrate. It was *Leu 6.91 ± 0.61 7.08 ± 0.01 5.9
observed that for both fractions, the limiting amino acids *Lys 9.49 ± 0.22 11.62 ± 0.43 4.5
were Thr and Trp. Additionally, similar to other legumes, *Met + Cys 2.04 ± 0.15 3.09 ± 0.01 2.3
globulins displayed a limitation in sulphur-containing amino *Tyr + Phe 4.65 ± 0.34 3.57 ± 0.07 4.1
acids (Met + Cys), while glutelins exhibited reduced con- *Thr 0.08 ± 0.04 0.06 ± 0.24 2.5
tents of aromatic amino acids (Tyr + Phe), whose levels do *Trp 0.31 ± 0.03 0.30 ± 0.45 0.6
not reach the recommendations by FAO/WHO (2007). The *Val 4.26 ± 0.01 3.99 ± 0.11 4.0
higher amount of essential amino acids in globulins and glu- Total EAA 36.32 ± 1.24 36.40 ± 1.26 28.5
telins compared to the recommended values suggests that LL Ala 3.69 ± 0.01 4.20 ± 0.02
seed proteins could serve as valuable protein sources, espe- Arg 11.72 ± 0.01 11.68 ± 0.05
cially in regions where protein deficiencies are prevalent. Asp 9.20 ± 0.21 9.19 ± 0.01
Besides, the presence of hydrophobic amino acids in globu- Asn 0.18 ± 0.80 0.28 ± 0.90
lins suggests these proteins may have improved solubility Glu 21.04 ± 0.01 23.55 ± 0.04
and emulsification capabilities, which might be useful in a Gln 0.08 ± 0.12 0.89 ± 0.01
variety of food and beverage applications. The deficiency Cys 1.29 ± 0.65 2.91 ± 0.43
in sulfur-containing amino acids in CT protein concentrates Ser 0.68 ± 0.04 0.86 ± 0.21
may be compensated by blending them with cereals or other Tyr 2.89 ± 0.10 1.95 ± 0.06
legumes rich in Met and Cys. This approach opens avenues Gly 4.15 ± 0.05 4.88 ± 0.01
leading to the formulation of balanced protein blends with Pro 5.56 ± 0.24 5.79 ± 0.15
improved nutritional profiles (Pastor-Cavada et al. 2014). Amino acids type distribution (%)
However, the total essential amino acid content in globulins Hydrophobic 41.40 ± 1.05 35.10 ± 1.07
(36.32%) and glutelins (36.40%) was higher than recom- Hydrophilic 58.60 ± 1.43 64.90 ± 1.33
mendations for adults (28.5%) (FAO/WHO 2007). Hydro-
Values are mean ± standard deviation of triplicate determinations
phobic amino acids were predominant in globulins (41.40%)
Hydrophobic (Ala, Val, Met, Phe, Leu, Ile, Pro, Trp, Gly, Tyr)
compared to glutelins (35.10%); these types of amino acids
Hydrophilic (Arg, Asp, His, Lys, Glu, Asn, Gln, Cys, Thr, Ser)
contribute to some functional properties such as solubility,
emulsification, and foaming (Foegeding 2015). *EAA Essential amino acids

Gel electrophoresis (SDS‑PAGE)
The electrophoretic profile of globulins (B) and glutelins
(C) protein fractions of CT are shown in Fig. 2. Defined
globulin fractions at ~ 50, 37, and ~ 23 kDa were found,
which correspond to 7S and 11S subunits. The 7S globu-
lins fraction is formed by convicilins (~ 76–80 kDa), and
vicilins (~ 50 kDa), and 11S is a hetero-hexamer formed
by acidic (~ 30–40 kDa) and basic (17–20 kDa) polypep-
tide bands. In the case of glutelins (30%), only one clear
band (~ 50 kDa) was observed, probably corresponding to
a vicilin-like fraction (7S globulin) (Tan‐Wilson and Wil-
son 2011). This analysis offers a glimpse into the structural
composition of LL seed proteins, indicating the presence
of characteristic globulin and glutelin fractions similar to
those found in other legume seeds. However, the protein
fractions profile for lupin bean seeds had shown bands at
similar molecular weights (~ 23, 37, and 50 kDa) attrib-
uted to the subunit constituents of legumin and vicilin
(Rodríguez-Ambriz et al. 2005). Comparing the electro-
phoretic profiles of LL seed proteins to those of other leg-
umes provides useful information for protein engineering
and crop development efforts.
The secondary structure of powdered globulins and glu-
telins fractions analyzed by FTIR spectra showed the typical
protein footprint (Fig. 3), providing insights into how protein
extraction conditions affect protein structure. Both fractions
exhibited a consistent pattern with variations in absorbance
intensity, the peaks associated with amide groups can be
seen around 3285 ­cm−1 (Amide A associated with N–H

Fig. 2  SDS-page analysis for globulins. (A) Protein standar marker,
(B) and glutelins (C) protein fractions from Leucaena leucocephala
seeds
stretching coupled with hydrogen bonding), 2922 ­cm−1
(Amide B, –CH stretching vibration), 1630 ­cm−1 (Amide I,
stretching vibrations of the C=O bond), 1513 ­cm−1 (Amide
II, N–H bending), and around 1237 ­cm−1 (Amide III, C–N
stretching and N–H bending). The distinct secondary struc-
ture patterns observed in globulins and glutelins within
the protein backbone suggest variations in their functional
attributes. The Amide I band, which represents secondary
structures in legume proteins, includes β-sheet, α-helix, ran-
dom coil, and β-turn. These structural components may vary
slightly due to differences in amino acid composition and
interactions among functional groups. Globulins with higher
amounts of α-helix and β-sheet structures may provide better
Fig. 3  FTIR spectra of globulins and glutelins protein fractions from
Leucaena leucocephala cotyledon
Chemical Papers
stability and solubility, making them useful for protein dis-
persion and emulsification. Understanding these structural
differences can help guide the selection and optimization of
extraction procedures and processing processes for tailoring
LL seed proteins to specific applications, hence increasing
their versatility. The FTIR spectrum reveals the abundance
of α-helix and β-sheet structures for globulins compared to
glutelins. In other sources of legumes (Phaseolus vulgaris,
Phaseolus calcaratus, Dolichos lablab), higher amounts of
β-sheet and relatively lower of α-helix have been reported
(Law et al. 2008; Rui et al. 2011).
Protein functionality
Protein solubility
The solubility of globulins and glutelins as a function of pH
(2–10 values) is shown in Fig. 4. Both fractions showed the
typical U-shaped profile. Similarly, globulins and glutelins
showed the lowest solubility values (3.03 and 3.36%, respec-
tively) at pH 6 (closer to the isoelectric point of the protein).
Decreasing the pH value to 2 enhanced the solubility of both
protein fractions, and it was observed that globulins were
significantly more soluble (~ 72%) than glutelins (~ 63%). A
similar behavior was observed at a pH of 10, where globulins
exhibited a higher solubility (37.34%) compared to 21.36%
for glutelins. This suggests that the solubility properties of
the two protein types differ in alkaline environments, with
globulins exhibiting higher solubility than glutelins. In that
sense, globulins exhibited higher solubility than glutelins,
particularly at acidic pH levels, indicating their suitabil-
ity for acidic formulations like juices, dairy products, and
soups. However, both fractions displayed lower solubility at
neutral pH, which may limit their use in neutral or slightly
alkaline products. The observed differences in solubility
between globulins and glutelins suggest opportunities for
Fig. 4  Solubility profile as a function of pH for globulins and glute-
lins fractions from Leucaena leucocephala protein cotyledon. Data
points represent means (n = 3) ± SD
Chemical Papers
formulating protein blends with customized solubility pro-
files to meet specific product needs. The protein solubility
has been related to the balance between protein–protein and
protein–solvent interactions. Also, it can be affected by pH,
ionic strength, temperature, solvent polarity, and processing
conditions (Shevkani et al. 2015). Solubility values for LL
protein were in agreement with solubility found for vici-
lin and legumin isolate from pea protein at the same pH
range (2–10). The lower solubility of the protein occurred
around pH between 5 and 6 and progressively increased far
away from these values; also, the highest solubility (> 80%)
occurred at pH 2 (Liang and Tang 2013). Solubility is an
important characteristic in food proteins when its intended
use is as an additive in mixed systems such as emulsions,
foams, or gels. Additionally, exploring processing tech-
niques like enzymatic hydrolysis or microencapsulation
could enhance the solubility and functionality of LL pro-
tein fractions, expanding their potential applications in the
food industry. In this case, the potential use of these protein
fractions could be in partially acid products like juices, dairy
products, and other semiliquid products like sauces, soups,
infant milk formulations, and ready-to-drink protein shakes
(Khazaei et al. 2019).
Emulsifying capacity (EC) and emulsion thermal
stability (ES)
Both EC and ES in LCP protein fractions varied with pH
changes, as depicted in Fig. 5. EC in glutelins at pH 4
(38.18%) was significantly higher (P < 0.05) than the maxi-
mum value for globulins (35.13%) at pH 2. Similarly, solu-
bility at a pH close to IEP, the EC decreased for glutelins
(7.14%) and globulins (11.76%) fractions. The behavior of
ES and EC was the same for both the fractions, with higher
values at acidic and alkaline pH values (> 95%) and lower
at pH 6 (11.76% and 7.14%, respectively). The EC results
of globulins were similar to findings reported by Sethi and
Kulkarni (1994b) in LL seed globulins, where EC (~ 40%)
was better when compared to soy protein (~ 30%). Other
legume proteins, such as Canavalia ensiformis protein iso-
lates, have shown similar behavior but higher EC (51–54%),
and in the case of Phaseolus lunatus, EC ranged around
41.78–56.42% (Chel-Guerrero et al. 2002). The superficial
net charges in a protein molecule can be enhanced by the pH
of the media and, thus, increase the water–protein interac-
tions (solubilization) and hydrophobic interactions required
for a protein to cover the oil droplet surface (Withana-
Gamage et al. 2011). During the application of mechani-
cal forces (high-speed homogenization), a probable partial
protein denaturation occurs, exposing hydrophobic groups
and favoring the non-polar interactions with the oily phase in
the system (Jones 2016). The observed variations in EC and
ES of LL protein fractions underscore the complex interplay
between protein structure and environmental factors such as
pH. The pH-dependent changes in EC highlight the dynamic
nature of protein interactions at different charge states, influ-
encing their ability to stabilize oil-in-water emulsions. The
higher EC exhibited by glutelins at pH 4 compared to globu-
lins at pH 2 suggests distinct pH optima for emulsification,
potentially due to differences in surface hydrophobicity and
charge distribution. Furthermore, the decline in EC near the
IEP (pH 6) for both fractions underscores the critical role
of electrostatic repulsions in emulsion stability, with impli-
cations for formulation strategies aimed at optimizing pH
conditions for enhanced emulsifying properties. Similarly,
the pH-dependent trends observed in ES reflect the delicate
balance between protein–protein and protein–oil interactions
in maintaining emulsion stability. The higher ES at acidic
and alkaline pH values (> 95%) suggests stronger protein–oil
interactions under these conditions, leading to improved
thermal stability of emulsions. Conversely, the reduced ES
near the IEP highlights the vulnerability of emulsions to
destabilization due to decreased repulsive forces between
protein molecules. These insights into the pH-dependent
behavior of LL protein fractions provide valuable guidance
for formulating emulsions with tailored stability profiles,
thereby expanding their potential applications in new food
development. Further investigation into the underlying
mechanisms governing pH-mediated changes in EC and ES
will facilitate the development of optimized emulsion sys-
tems with superior stability and functionality.
Foaming capacity (FC) and foaming stability (FS)
The pH differences of strong acid or alkaline solutions can
contribute to protein structure unfolding and increase the
flexibility of the polypeptide chains, facilitating its diffusion
to the air–water interface (Raikos et al. 2014). The FC and
FS of LL protein fractions as a function of pH are shown in
Fig. 6. The FC of globulins was higher at pH 2 (~ 179%),
unlike glutelins at pH 10 (~ 135%). For both samples, the
lowest FC was at pH 6 (~ 132 and 83%, respectively). At pH
values distant from an IEP, the FC improved for all samples.
The absence of charges on protein surface at IEP could cause
the reduction of interactions at the interface (water–air) or
protein precipitation and thus, air bubbles disruption. The
FS for globulins was better compared to glutelins at almost
all pH values (except pH 10), and contrary to FC, the FS was
enhanced near the IEP (pH 8), showing the highest stability
(98.01%). Different phenomena drive foam formation and its
mechanisms of stabilization; in the case of FS, it is required
the formation of a stable molecular layer at the air–water
interface. It has been shown that a low superficial net charge
in proteins close to the IEP, promotes a more stable layer
in this region (Karaca et al. 2011). Some previous reports
have indicated that proteins exhibit increased FS, a property
 Chemical Papers

food formulations. The higher FC of globulins at acidic pH

and glutelins at alkaline pH suggests their suitability for
different types of foaming applications. Globulins may be
preferred for acidic food products like fruit-based desserts
or beverages, where stable foaming is desirable, while glu-
telins could be more suitable for alkaline products such as
plant-based meat alternatives or dairy substitutes. Within the
protein family, albumins stand out for their notable structural
and functional diversity. The higher FS of LL protein frac-
tions near the IEP suggests the formation of a more stable
molecular layer at the air–water interface, enhancing foam
stability. This property is crucial for applications requiring
long-term foam stability, such as whipped toppings or aer-
ated confections. Specifically, the FS of albumins derived
from lentils (Lens culinaris) and horse gram (Macrotyloma
uniflorum) is significantly higher, with values of approxi-
mately 66 and 55%, respectively, in contrast to their globulin
counterparts, which show comparatively lower FS percent-
ages of 6.7 and 4.4%, respectively. These findings highlight
the intricate interplay among protein structure, source, and
observed functional properties, underscoring the signifi-
cance of considering distinct protein fractions when eval-
uating characteristics such as FS (Ghumman et al. 2016).
Comparisons with other legume-derived albumins further
highlight the unique functional attributes of LL protein
fractions, emphasizing the need to consider both physico-
chemical and structural characteristics when evaluating their
foaming properties. By leveraging these insights, researchers
and formulators can tailor LL protein fractions for specific
Fig. 5  Emulsifying capacity (a) and thermal stability (b) as a func- foaming applications, contributing to the development of
tion of pH for globulins and glutelins fractions from Leucaena leuco-
innovative food products.
cephala protein cotyledon. Data points represent means (n = 3) ± SD

Water holding capacity (WHC) and oil absorption

attributed to their functional attributes directly linked to both capacity (OAC)
physicochemical and structural characteristics. The varia-
tion in FC and FS of LL protein fractions across different In addition to their structural differences, the contrasting
pH levels offers insights into their potential applications in WHC and OAC between globulins and glutelins highlight

a) Globulins b)
a a
180 Glutelins 100 a a
a
b
a a
a a
Foaming capacity (%)

b
Foaming stability (%)

a a
a a 80 b
b a b a
b
120 b b
b 60 Globulins
b b b
b 120 min
40
60 Glutelins
20
120 min
0 0
2.0 2.3 3.0 4.0 6.0 8.0 10.0 2.0 2.3 3.0 4.0 6.0 8.0 10.0
pH pH

Fig. 6  Foaming capacity (a) and foaming stability (b) as a function of nificant difference (Tukey's HSD test, P < 0.05), among globulins and
pH for globulins and glutelin fractions from Leucaena leucocephala glutelin fractions. Data points represent means (n = 3) ± SD
protein cotyledon. Different letters on the column top indicate the sig-
Chemical Papers
a)
Oil absorption capacity g /g protein)
Water holding capacity (g /g protein)
5 4
a a
b
4 extraction and processing methods to tailor LL protein frac-
3 b
3
2
2
1
1 Conclusions
0 0
Globulins Glutelins
Protein fraction
Fig. 7  Water holding (a) and oil absorption (b) capacity for globulins
and glutelin fractions concentrates from Leucaena leucocephala seed
cotyledon. Different letters on the column top indicate the significant
difference (Tukey's HSD test, P < 0.05), among globulins and glutelin
fractions. Data points represent means (n = 3) ± SD
their potential applications in food development. In Fig. 7,
the results of the WHC and OAC of LL CT proteins are
shown. Both WHC and OAC were significantly higher
(P < 0.05) for globulins than glutelins (4.17–3.25 and
3.86–2.58 g/g, respectively). The results of WHC for both
globulins and glutelins were higher than values reported for
protein isolated from LL (1.50 g/g) by Sethi and Kulkarni
(1994b), pea protein isolates (0.3–2.6 g/g) (Stone et al.
2015), and by WHC values for soybean, black soybean,
Mung bean (Vigna angularis), and Adzuki bean (Vigna
angularis) protein isolates with a 3.76, 4.27, 4.09, and
3.75 g/g, respectively (Chen et al. 2017). The structural dif-
ferences between protein fractions are distinctly contribut-
ing to their behavior. According to the amino acid profile,
the amount of hydrophobic amino acids in globulins was
higher (~ 41%) than in glutelins (~ 35%). The OAC in this
study was lower than LL protein isolates (4.50 g/g) by Sethi
and Kulkarni (1994b), but similar to Canavalia ensiformis
protein isolate (2.70 g/g) (Chel-Guerrero et al. 2002). Nev-
ertheless, higher values were found compared to Phaseolus
angularis (1.28 g/g) and commercial soy protein isolates
(1.39 g/g) (Meng and Ma 2002). The OAC in food products
is an important functional property because it is useful in
structural interactions in food, especially in flavor retention,
improvement of palatability, and extension of shelf life in
meat products by reducing moisture and fat loss (Kinsella
1979). Additionally, external factors (stirring speed, pH, and
protein concentration) that can be changed during recovery
or measurement can also influence this property. Herein,
globulins, with their higher WHC and OAC, may be more
suitable for applications requiring moisture retention and
flavor absorption, such as meat analogs and bakery products.
On the other hand, the lower WHC and OAC of glutelins
may make them more suitable for applications where oil
retention is undesirable, such as in low-fat food formula-
tions or oil-free products. In addition, deeper studies into
b) the parameters that influence WHC and OAC, such as pH,
stirring speed, and protein concentration, can help optimize
tions for specific uses.
The results of this research support the idea that the underu-
Globulins Glutelins
tilized LL biomass is a potential sustainable source of high
Protein fraction
nutritional and low-cost food ingredients. Also, lays the
groundwork for future research into bioactive phytochemi-
cals from LL biomass. The protein content and functional-
ity of protein fractions from its seeds CT provide insights
for desirable functional properties at a wide pH range and
thermal stability. Overall, this study strengthens the idea
that cultivation of this crop could be incentivized to produce
added-value food additives in developing more affordable
products for low-income population sectors.
Acknowledgements This work was financially supported by Departa-
mento Regional de Bioingenierías, Tecnológico de Monterrey, Queré-
taro, México and Consejo Nacional de Ciencia y Tecnología (CONA-
CYT) for first author scholarship (No. 305253). The authors also want
to thank project CONACYT (FOINS 4950) for reagents support.
Declarations
Conflict of interest The authors declare that no conflict of interest ex-
ists.
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