Chia expeller: A promising source of antioxidant, antihypertensive and antithrombotic peptides produced by enzymatic hydrolysis with Alcalase and Flavourzyme

Abstract

Chia expeller is a promising source of bioactive compounds suitable for the development of nutraceutical ingredients due to its functional, biological, and nutritional properties. In this work, chia expeller was hydrolysed with Alcalase-Flavourzyme sequential system and compared to the individual enzymes. A higher degree of hydrolysis (57.63 ± 6.08%) was obtained after 90 min-Alcalase and 90 min-Flavourzyme (H-A90-F90), with the development of low molecular weight peptides as observed by SDS-PAGE. H-A90-F90 exhibited antiradical activity with ABTS (TE=4.87 ± 0.13 mmol L⁻¹ mg⁻¹), DPPH (TE=1.55 ± 0.02 mmol L⁻¹ mg⁻¹), antihypertensive activity (45% ACE-I inhibition), and antithrombotic activity against both intrinsic and extrinsic coagulation pathways. These results represent the first report of antithrombotic peptides from Salvia hispanica, highlighting the relevant use of chia seed by-products to obtain potentially antioxidant, antihypertensive, and anticoagulant peptides by enzymatic hydrolysis with Alcalase and Flavourzyme, enhancing this agro-industrial by-product.

Full text

Food Chemistry 380 (2022) 132185
Available online 19 January 2022
0308-8146/© 2022 Elsevier Ltd. All rights reserved.
Chia expeller: A promising source of antioxidant, antihypertensive and
antithrombotic peptides produced by enzymatic hydrolysis with Alcalase
and Flavourzyme
Brenda Oz´
on
a
,
1
, Juliana Cotabarren
a
,
1
,
*
, Tania Valicenti
b
, M´
onica Graciela Parisi
b
,
Walter David Obreg´
on
a
,
*
a
Centro de Investigaci´
on de Proteínas Vegetales (CIProVe), Departamento de Ciencias Biol´
ogicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 47 y
115 s/N, B1900AVW, La Plata, Argentina
b
Laboratorio de Química Biol´
ogica, Departamento de Ciencias B´
asicas, Universidad Nacional de Luj´
an. Ruta 5 y Avenida Constituci´
on, Luj´
an, 6700 Buenos Aires,
Argentina
ARTICLE INFO
Keywords:
Salvia hispanica L.
Enzymatic hydrolysates
Chia by-products
Expeller
Sequential hydrolysis
Antioxidant compounds
Antithrombotic peptides
Antihypertensive peptides
ABSTRACT
Chia expeller is a promising source of bioactive compounds suitable for the development of nutraceutical in-
gredients due to its functional, biological, and nutritional properties. In this work, chia expeller was hydrolysed
with Alcalase-Flavourzyme sequential system and compared to the individual enzymes. A higher degree of hy-
drolysis (57.63 ±6.08%) was obtained after 90 min-Alcalase and 90 min-Flavourzyme (H-A90-F90), with the
development of low molecular weight peptides as observed by SDS-PAGE. H-A90-F90 exhibited antiradical ac-
tivity with ABTS (TE =4.87 ±0.13 mmol L
-1
mg
−1
), DPPH (TE =1.55 ±0.02 mmol L
-1
mg
−1
), antihypertensive
activity (45% ACE-I inhibition), and antithrombotic activity against both intrinsic and extrinsic coagulation
pathways. These results represent the rst report of antithrombotic peptides from Salvia hispanica, highlighting
the relevant use of chia seed by-products to obtain potentially antioxidant, antihypertensive, and anticoagulant
peptides by enzymatic hydrolysis with Alcalase and Flavourzyme, enhancing this agro-industrial by-product.
1. Introduction
Chia (Salvia hispanica L.) is a well-known annual herbaceous plant
that belongs to the Lamiaceae family, whose production, consumption,
and demand have strongly increased in recent years because of its
interesting nutritional prole (Ayerza & Coates, 2011; Ixtaina, 2010).
Native to Mesoamerica, it was one of the main components of the Aztec
diet, also used as a medicine and food supplements (Mu˜
noz et al., 2013).
Chia began to be internationally commercialized in the nineties for
its healthy and functional properties, moving around 50 billion dollars
per year in the world, a number that is increasing. Its main producers
-Mexico, Bolivia, Paraguay, Argentina, Ecuador, Nicaragua, Guatemala,
and Australia- are expanding export markets previously dominated by
the United States, to European Nations and other countries such as
Canada, China, Malaysia, Singapore and the Philippines since its in-
clusion in processed foods was authorized (Busilacchi et al., 2015;
Kulczynski et al., 2019).
There is currently much concern in the agri-food sector about envi-
ronmental problems caused by the disposal of waste and by-products.
Oil industries, particularly chia oil, produce around 650 kg of chia
expeller/ton. Flours obtained from oilseeds (also called expellers), by-
products of edible oil industries after oil extraction, are under-
estimated as sources of protein for human consumption, often discarded
or conventionally used as animal feed, in energy production, in cosmetic
and cosmeceutical, as biomaterials or as fertilizer (Vinayashree & Vasu,
2021). That is why several strategies are being developed to reduce
waste and losses resulting from activities carried out in the food system.
An interesting approach consists in increasing the added value of these
agro-industrial wastes, through the use or recycling of these by-
products, thus contributing to a sustainable circular bioeconomy and
the protection of the environment. Therefore, the chia expeller provided
by local industries has been gaining popularity as a promising source of
bioactive peptides in the development of nutraceutical formulations and
as functional foods due to its biological, functional and nutritional
* Corresponding authors.
E-mail addresses: cotabarren.juliana@biol.unlp.edu.ar (J. Cotabarren), davidobregon@biol.unlp.edu.ar (W. David Obreg´
on).
1
These authors contributed equally to this work.
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
https://doi.org/10.1016/j.foodchem.2022.132185
Received 30 June 2021; Received in revised form 23 December 2021; Accepted 15 January 2022

Food Chemistry 380 (2022) 132185
2
properties.
Bioactive peptides exert important benecial effects for health
because they have multiple potential biological activities such as anti-
oxidant, antihypertensive, antithrombotic, antimicrobial, anticancer,
immunomodulatory, and anti-inammatory among others. They are an
alternative for the prevention of different metabolic diseases, due to
their wide spectrum of action, biospecicity, low allergenicity, and
because they are rapidly degraded in the environment (Cruz-Casas et al.,
2021), The World Health Organization (WHO) has emphasized on the
rational use of natural medicines in the treatment of prevalent diseases
(WHO, 2003). Cardiovascular diseases (CVDs) are the most prevalent
cause of deaths worldwide, directly related to atherosclerosis and its
well-established risk factors. Thrombi (blood clots) are known to cause
heart attacks if they lodge in the coronary artery, so the design of
functional foods with antioxidant, antihypertensive and antithrombotic
peptides could be a natural and interesting strategy to reduce the inci-
dence of these pathologies. Chakrabarti et al. (2014) studied the bio-
logical importance of antioxidant peptides, to improve the nutraceutical
and functional framework of food by increasing biological defence
mechanisms against inammatory diseases due to oxidative stress. On
the other hand, Cicero et al. (2017) emphasized the multivariate activ-
ities of BAPs and their impact on heart-related diseases, being respon-
sible for the main causes of death in the world. Despite the importance of
the search for antithrombotic peptides of natural origin, no previous
studies have been carried out on the presence of this activity on chia
expeller.
The main objective of this work is to highlight the relevant
employment of chia seed by-products in the obtaining of potentially
antioxidant, antihypertensive, and antithrombotic peptides from chia
proteins hydrolysed with Alcalase, Flavourzyme and sequentially with
Alcalase and Flavourzyme, encouraging the valorisation of this agro-
industrial by-product.
2. Materials and methods
2.1. Materials and reagents
Protease from Bacillus licheniformis (Alcalase), Protease from Asper-
gillus oryzae (Flavourzyme), sodium chloride, tris (hydroxymethyl)
aminomethane, sodium dodecyl sulphate (SDS), β-mercaptoethanol
(βME), Coomassie Blue G-250, N,N,N′,N′-tetramethyl ethylene diamine
(TEMED), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-Azino-bis(3-eth-
ylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), (±)-6-Hy-
droxy-2,5,7,8-tetramethylchromane-2-carboxylicacid (Trolox), o-
phthaldialdehyde (OPA), Angiotensin Converting Enzyme from rabbit
lung, Hippuryl-Histidyl-Leucine and bovine serum albumin were sup-
plied by Sigma-Aldrich (U.S.A.).
2.2. Crude extract
Chia expeller (6 to 8% of residual oil content) was provided by
GREENBORG S.R.L. and used as starting material. The defatting of the
expeller was carried out with ethanol (85%) with shaking at 200 rpm for
3 h according to Cotabarren et al. (2019), and after that, it was grinded
to produce a our with high protein content. The defatted our was
analysed to determine dry matter (stove 105 ◦C for 4 h), crude protein
(Kjeldahl method) and fat content by Soxhlet method. Chia protein
concentrate was obtained by mixing 40 g of defatted our with 1000 mL
of 0.01 M phosphate buffer, 0.1 M NaCl, pH 7.4 and homogenizing into a
cool blender. The mixture was stirred for 1 h at room temperature,
ltered with gauze, and the suspension was then claried by centrifu-
gation (7,000 ×g for 1 h at 4 ◦C). The supernatant called “crude extract”
(CE) was stored at −20 ◦C until analysis.
2.3. Enzymatic hydrolysis
Enzymatic hydrolysis of the CE was performed with the two food-
grade microbial proteases Alcalase (enzyme/substrate ratio of 0.3 U
g
−1
) and Flavourzyme (enzyme/substrate ratio of 50 U g
−1
) separately.
The reaction mixture was adjusted to pH 7.0 to achieve the optimal
enzymatic activity, incubated for 3 h at 50 ◦C and 80 rpm in a shaker.
Samples were withdrawn in duplicate at the selected time intervals (see
Table 1), and hydrolysis was stopped by heating at 85 ◦C for 15 min. The
insoluble material was removed by centrifugation at 13,000 ×g for 30
min and 4 ◦C. The hydrolysates were then lyophilized and kept at
−20 ◦C.
As described in Table 1, for the sequential hydrolysis with both en-
zymes (Alcalase-Flavourzyme), the CE was subjected to 90 min of hy-
drolysis with Alcalase followed by increasing times with Flavourzyme,
in compliance with the conditions as previously demonstrated. The
sample control (from now on: C) was obtained by subjecting the CE to
the same hydrolysis conditions without the addition of the enzymes.
2.4. Characterization of hydrolysates
2.4.1. Determination of the degree of hydrolysis
The degree of hydrolysis (DH%) was quantied with the o-phtha-
laldehyde method according to Nielsen et al. (2001). The OPA reagent
was prepared by completely dissolving 0.4767 g di-Na-tetraborate
decahydrate and 125 mg Na-dodecyl-sulphate (SDS) in 12 mL deion-
ized water. The OPA solution was carried out by combining 10 mg of o-
phthaldialdehyde 97% (OPA) in 250 µL methanol and then transferred
quantitatively to the above-mentioned solution with the addition of 25
µL β-mercaptoethanol, by rinsing with deionized water. Finally, the
solution was made up to 13 mL with deionized water. The assay was
carried out by adding 10 µL sample to 200 µL of OPA reagent by trip-
licate. After 2 min-incubation, the absorbance at 340 nm was measured
using a using a Tecan Innite M200 PRO spectrophotometer
(M¨
annedorf, Switzerland). The percentage of DH was calculated using
the following formula:
DH% =h/h
tot
×100;where h is the number of hydrolysed bonds and
h
tot
is the total number of peptide bonds per protein equivalent which
depends on the amino acid composition of the raw material. 100% of
hydrolysis was determined by complete hydrolysis of the protein with 6
N HCl at 100 ◦C for 24 h.
2.4.2. SDS-Tricine-PAGE
Molecular mass proles of the different hydrolysates were charac-
terized by SDS-Tricine-PAGE according to Sch¨
agger (2006). A sample of
30 µg of protein of each fraction was mixed with sample buffer (Tris
0.13 M, SDS 2%, β-mercaptoethanol 5% v/v, glycerol 8% v/v, bromo-
phenol blue 0.002% p/v, pH 6.8) and incubated 5 min at 100 ◦C. Then,
samples were subjected to denaturing and reducing electrophoresis at a
constant current of 15 mA per gel using a Mini – Protean III dual slab cell
(Bio-Rad, Hercules, CA 94547, USA), using 4% stacking gel and 16%
separating gel. After electrophoresis, the gels were stained with 0.2%
Coomassie Brilliant Blue G-250 and destained with distilled water.
Table 1
Hydrolysis conditions used to obtain chia expeller protein hydrolysates.
Enzyme Sample
Denomination
Enzyme/
Protein (U
g
¡1
)
pH T
(◦C)
Time
(min)
Alcalase H-A 0.3 7 50 0–240
Flavourzyme H-F 50 8 50 0–240
Alcalase-
Flavourzyme
H-A-F 0.3
50
7
8
50
50
90
0–240
B. Oz´
on et al.

Food Chemistry 380 (2022) 132185
3
2.4.3. Size exclusion chromatography
A 1 mL aliquot of the selected hydrolysate (H-A90-F90) and control
(C) was loaded onto a Sephacryl S100 HR column (1.6 ×40 cm) con-
nected to an ¨
Akta-Purier (GE Healthcare) previously equilibrated with
milliQ water. Elution was carried out at a ow rate of 0.8 mL min
−1
monitoring 215 nm absorbance.
A 2 mL aliquot of H-A90-F90 and C was loaded onto a Superdex 30
prep grade column (1.6 ×80 cm) connected to an ¨
Akta-Purier (GE
Healthcare) previously equilibrated with milliQ water. Elution was
carried out at a ow rate of 0.4 mL min
−1
monitoring 215 nm
absorbance.
2.5. Antioxidant activity assays
2.5.1. DPPH radical scavenging activity
The scavenging activity against DPPH free radicals was measured
according to Brand-Williams et al. (1995) with slight modications and
adapted to a 96-well at bottom plate (Cotabarren et al., 2019).
Briey, samples (50
μ
L) were mixed with 50
μ
L of 0.2 mmol L
-1
DPPH
radical solution dissolved in 150 µL methanol. The maximum DPPH
absorbance was measured by replacing the sample volume with water.
After 20 min incubation at 37 ◦C in dark conditions, absorbance was
measured at 517 nm. Measurements were carried out in triplicate.
DPPH radical scavenging activity was calculated using the following
formula:
DPPHradicalscavengingactivity(%) = 100 ×A1−A2
A1
where A1 is the absorbance of the control without sample and A2 is the
absorbance in presence of sample and DPPH.
The reaction was performed for six different concentrations for each
sample and the IC
50
value was dened as the antioxidant compound
concentration required to scavenge 50% of the DPPH radical.
2.5.2. ABTS
+
radical scavenging activity
The ABTS
+
radical scavenging activity was determined according to
Pukalskas et al. (2002) with slight modications and adapted to a 96-
well at bottom plate. The ABTS
•+
radical cation was produced by
reacting ABTS with potassium persulfate in a 2:1 ratio and dissolved in 5
mL of distilled water, allowing the mixture to stand in darkness at room
temperature for 16–17 h before use. The resulting blue-green ABTS
+
solution was diluted with phosphate buffer pH 7.5 until its absorbance
reached 1 ±0.01 AU at 734 nm.
Antioxidant compound content in the crude extract and selected
hydrolysate was analysed adding 190
μ
L of diluted ABTS
•+
solution to
10
μ
L sample or Trolox standard (nal concentration 0–100 µM). After
10 min incubation at room temperature in dark conditions, absorbance
was measured at 734 nm. Six different concentrations of each sample
were performed, and measurements were carried out in triplicate.
The percentage decrease in absorbance at 734 nm was estimated and
plotted as a function of the Trolox concentration for the standard
reference data and expressed as mmol of Trolox equivalents (TE) per mg
of protein.
ABTS
+
radical scavenging activity was calculated using the following
formula:
ABTSradicalscavengingactivity(%) = 100 −100 ×A1−A2
A0
where A0 is the absorbance of the control without sample, A1 is the
absorbance in presence of the sample and ABTS
•+
, and A2 is the
absorbance of sample blank without ABTS
•+
.
IC
50
value was dened as the antioxidant compound concentration
required to scavenge 50% of the ABTS radical.
2.6. ACE-inhibitory activity
The ACE inhibitory activity was measured according to the method
described by Chang et al. (2001) which is based on a selective chro-
mogenic reaction for histidyl-leucine (λ
max
=390 nm) using o-phtha-
laldehyde. Twenty-ve microliters of sample (1.2 mg mL
-1
C and H-A90-
F90) were pre-incubated with 50
μ
L of substrate (4.7 mM hippuryl-His-
Leu in 0.1 M borate buffer pH 8.3, containing 0.3 M NaCl) at 37 ◦C. Then
50
μ
L of 25 mU mL
−1
ACE solution were added followed by an incu-
bation at 37 ◦C for 40 min. Aliquots (25
μ
L) of the reaction mixture were
added into each well containing 150
μ
L of 0.3 M NaOH and 10
μ
L of 2%
o-phthalaldehyde in methanol on a 96-well microplate. Finally, the re-
action was terminated by adding 20
μ
L of 3 M HCl and kept at room
temperature for 10 min. The absorbance generated was recorded at 390
nm, in a Tecan Innite M200 PRO spectrophotometer (M¨
annedorf,
Switzerland). For the control and blank, water was added instead of
samples and ACE, respectively. The inhibition rate was calculated as the
following:
ACEinhibitoryactivity(%) = (1−As −Ab
Ac −Ab)×100
where As is the absorbance of the test sample in the presence of the
reaction mixture, Ab is the absorbance of test sample in the absence of
ACE, and Ac is the absorbance of buffer in the absence of the test sample.
Each sample was analysed in three technical repeats.
2.7. Blood coagulation assays
Antithrombotic activities of the selected hydrolysate (H-A90-F90)
and control (C) were evaluated by determining prothrombin time (PT)
and the time of activated partial thromboplastin (aPTT) using a Coatron
M1 coagulometer (TECO, Germany) according to Cotabarren et al.
(2020).
In both cases, a pool of human blood plasma (PBP) –collected from
the bloods supernatant after 3000 rpm centrifugation for 15 min– from
the mixture, in equal parts, of 5 healthy individuals, maintained at 37 ◦C
with 3.8% sodium citrate (sample:anticoagulant ratio 9:1) was used as a
sample.
For the PT test equal parts of the PBP and sample (H-A90-F90: 0–800
μ
g mL
−1
; C: 0–2000
μ
g mL
−1
) were incubated for 2 min at 37 ◦C, then
Soluplastin reactive (Wiener Lab.) was added and checked for the
coagulation time. For the aPTT test, aPTT reactive (Wiener Lab.) was
added to an equal volume of PBP-sample mixture and, after 2 min in-
cubation at 37 ◦C, 25
μ
L of 50 mM CaCl
2
were added to initiate the
coagulation time determination. For both assays, measurements were
carried out in triplicate and appropriate controls were achieved.
2.8. Statistical analysis
Statistical analyses (ANOVA) were performed with GraphPad Prism
(v6.03, GraphPad Software Inc.: San Diego, CA, USA, 2012). All exper-
iments were conducted at least in triplicate. Data are expressed as the
mean ±standard deviation, and signicant difference was determined
by Tukey’s post-hoc test (p <0.05).
3. Results and discussion
3.1. Enzymatic hydrolysis of chia expeller
Chia expeller, a by-product of the oil extruded-pressing process was
provided by Greenborg Industries S.R.L. and defatted by solvent
extraction with ethanol (85%), achieving a protein and fat content of
36.8% and 0.88% respectively (Cotabarren et al. 2019), thus repre-
senting a good starter material for enzymatic hydrolysis.
Since the production of extensive hydrolysates (i. e., greater than
B. Oz´
on et al.

Food Chemistry 380 (2022) 132185
4
50% degree of hydrolysis – DH) requires the use of more than one
protease, in the present study an Alcalase-Flavourzyme sequential sys-
tem was used and compared to individual Alcalase and Flavourzyme
enzymes. These proteases were selected since both food-grade enzymes
of microbial origin are easy to purify with a lower production cost than
proteinase K and Chymotrypsin C enzymes, being suitable for use in
industrial processes (He et al., 2012).
Alcalase is a bacterial endoprotease that is limited by its specicity,
resulting in DHs no higher than 20–25% depending on the substrate,
although achieving these DHs in a relatively short time under moderate
conditions. It is known that this enzyme primarily hydrolyses peptide
bonds containing hydrophobic residues on the carboxyl side (Adler-
nissen, 1986), so starting materials rich in hydrophobic amino acids are
more suitable for Alcalase hydrolysis. Chim-Chi et al. (2018) demon-
strated that defatted chia our showed a predominant content of hy-
drophobic amino acids (42.87% w/w), followed by hydrophilic (41.24%
w/w) and neutral amino acids (15.89% w/w), being suitable for using
Alcalase in enzymatic hydrolysis.
As shown in Fig. 1 A, the highest degree of hydrolysis for Alcalase
occurred in the rst 90 min of hydrolysis (DH: 25.91 ±0.26%), with a
slight increase at times up to 240 min (DH: 32.09 ±4.11%). Similar
results (DH:19.88%) were reported by L´
opez-García et al. (2019) in the
hydrolysis of a protein-rich fraction obtained from defatted chia our
after 120 min of hydrolysis, while Coelho et al. (2018) showed a DH of
20.32 ±0.27% after 240 min of hydrolysis with the same starting
material.
Urbizo-Reyes et al. (2019) showed a DH of 33.64 ±1.44% when
hydrolysis was performed with Alcalase for 60 min using chia seeds as
starting material, while San Pablo-Osorio et al. (2019) observed a DH of
38.0% after 180 min of hydrolysis with the same starting material. Ac-
cording to these results, although the starting material used in this work
came from a by-product of the oil industry, the DH obtained was not
signicantly modied compared to that obtained by other authors using
the protein fraction from seed or defatted our, indicating that the chia
expeller maintains the protein quality of the seed.
When enzymatic hydrolysis was performed with Flavourzyme, a
maximum DH of 15.71 ±3.38% was observed at 120 min of reaction
(Fig. 1, A), after which there was no signicant increase. Similar results
were reported by Coelho et al. (2018) and by Zhao et al. (2012) since
they obtained a lower DH for Flavourzyme compared to those obtained
for Alcalase, which accounts for the cleavage specicity of the enzyme
used, suggesting that the peptide bonds of the native proteins in the
expeller are more susceptible to enzymatic hydrolysis with Alcalase.
As mentioned above, hydrolysates with high DH were produced
using a sequential enzyme system. Thus, taking into account the char-
acteristics of each enzyme, the initial use of Alcalase with its unique
endoprotease activity, allows to generate a wide variety of peptides,
while the use of Flavourzyme as the nal enzyme, favours the increase of
the DH due to its double endo and exoprotease activity. In this work, the
highest DH of 57.63 ±6.08% was observed after performing a sequen-
tial hydrolysis with Alcalase for 90 min followed by Flavourzyme for 90
min (H-A90-F90), after which the DH did not increase signicatively
(Fig. 1, B). These results are consistent with those reported by Segura-
Campos et al. (2013), who obtained a DH of 43.8% in a sequential hy-
drolysis with Alcalase (60 min) followed by Flavourzyme (150 min),
using chia seeds as starting material. Sosa Crespo et al. (2018). also
reported a DH of 45.7% after hydrolysing for 45 min with Alcalase
followed by Flavourzyme, while Urbizo-Reyes et al. (2019) reported a
DH of 46.81% in the same conditions.
SDS-PAGE (16%) was performed to analyse the protein prole of the
hydrolysates, nding that proteins with molecular weight higher than
20 kDa were fully digested after hydrolysing for 30 min with Alcalase
(Fig. 2, A). At longer hydrolysis times no changes in the prole were
observed. In addition, a decrease in the intensity of the protein prole
with molecular weight lower than 20 kDa was observed, being consis-
tent with the higher DH obtained at longer times. Furthermore, the
presence of low molecular weight bands that were not present in the
unhydrolyzed sample was evidenced (Bands I).
In the hydrolysates obtained with Flavourzyme (Fig. 2, B), only the
disappearance of proteins of 40–60 kDa (Bands II) was observed, while a
decrease in the intensity of proteins around 30 kDa and no differences
were observed for low molecular weight proteins.
Finally, for the hydrolysates produced by Alcalase-Flavourzyme
sequential system (Fig. 2, C), a decrease in the intensity of the low
molecular weight bands was observed only for the hydrolysate of 90
min-Alcalase and 30 min-Flavourzyme (H-A90-F30) (Bands III). No
differences were observed in the intensity and resolution of the bands
with molecular weight<20 kDa at longer hydrolysis times with Fla-
vourzyme. However, the results obtained by determining DH at a time
greater than 30 min indicate that Flavourzyme continues to generate
hydrolysis (Fig. 2, B), favouring the production of peptides of low mo-
lecular weight. According to these results, the sequential system H-A90-
F90 was selected for further analysis.
Differences were also observed in the protein prole of the control
(C) and the selected hydrolysate (H-A90-F90) in the gel ltration
chromatography with Sephacryl S100 HR and with the Superdex 30
prep grade, since the rst column allows to resolve proteins between 100
and 1 kDa, while the second allows the resolution of proteins between 10
and 0.5 kDa. As can be seen in Fig. 3, the chromatograms for both col-
umns showed signicant differences in absorbance when comparing the
control with the hydrolysed H-A90-F90. A considerable decrease in the
absorbance corresponding to the high molecular weight proteins of the
hydrolysate was observed compared to the control (Fig. 3, I), while the
absorbance of the peptides resolved at higher elution volumes increased
(Fig. 3, II). From the analysis of the gel ltration chromatography with
Superdex 30 prep grade, a higher resolution was observed for the low
Fig. 1. Degree of hydrolysis estimated by OPA. (A) Degree of hydrolysis of chia expeller using individual Alcalase (H-A) or Flavourzyme (H-F). (B) Degree of hy-
drolysis of chia expeller using sequential hydrolysis with Alcalase followed by Flavourzyme (H-A90-F). Each bar represents the mean and standard deviation of three
determinations. Bars labeled with different letters are signicantly different (p <0.05, one-way ANOVA and Tukeys multiple comparison test).
B. Oz´
on et al.

Food Chemistry 380 (2022) 132185
5
molecular weight peptides, since only 3 peaks with lower absorbance
were observed for the control while for the hydrolysate H-A90-F90, a
higher number of peaks with higher absorbance values were observed in
the same elution volumes. Thus, it can be concluded that the sequential
hydrolysis process with Alcalase and Flavourzyme favoured the pro-
duction of low molecular weight peptides with a decrease in complex
proteins.
3.2. In vitro biological activities
Bioactive peptides in foods are valuable functional agents in healthy
diets that can prevent and treat diseases. Since bioactive peptides are
encrypted in food, the degree of protein hydrolysis achieved in their
production is an important factor worth considering (Daliri et al., 2017).
Gilmartin & Jervis (2002) reported that protein hydrolysates produced
with combinations of enzymes or by sequential hydrolysis, can be used
to manipulate the molecular weight distribution and free amino acid
content, thus increasing the yields of specic hydrolysates containing
potentially bioactive low molecular weight peptides. These small pep-
tides contribute considerably to the biological potential of a protein
hydrolysate and are good candidates as in vivo physiologically bioactive
agents. Thus, the antioxidant, antihypertensive and antithrombotic ac-
tivities of the chia hydrolysates produced with the highest DH value (H-
A90-F90) were evaluated.
3.2.1. Antioxidant activity
Diseases related to oxidative stress and food quality deterioration are
of major concern worldwide, not only in terms of public health but also
in food production and conservation. Antioxidant peptides from food
proteins can be explored as new natural food ingredients with potential
therapeutic properties (Lorenzo et al., 2018).
In this work, the antioxidant activity was quantied and expressed as
Fig. 2. SDS-PAGE of hydrolysates with Alcalase
(A), Flavourzyme (B) and sequential Alcalase-
Flavourzyme (C). (A) Lanes: 1, molecular weight
marker; 2, control; 3, H-A30; 4, H-A60; 5, H-A90;
6, H-A120; 7, H-A150; 8, H-A180; 9, H-A210; 10,
H-A240. (B) Lanes: 1, molecular weight marker;
2, control; 3, H-F30; 4, H-F60; 5, H-F90; 6, H-
F120; 7, H-F150; 8, H-F180; 9, H-F210; 10, H-
F240. (C) Lanes: 1, molecular weight marker; 2,
control; 3, H-A90; 4, H-A90-F30; 5, H-A90-F60; 6,
H-A90-F90; 7, H-A90-F120; 8, H-A90-F150; 9, H-
A90-F180; 10, H-A90-F210; 11, H-A90-F240.
Fig. 3. Size-exclusion chromatography elution prole of the 90 min-Alcalase and 90 min Flavourzyme hydrolysate on Sephacryl S100 HR column (A) and Superdex
30 prep grade column (B). C: control; H-A90-F90: 90 min-Alcalase and 90 min-Flavourzyme hydrolysates.
B. Oz´
on et al.

Food Chemistry 380 (2022) 132185
6
Trolox equivalents (TE, mmol L
-1
mg
−1
) and as the concentration of
peptides that produces a 50% antioxidant activity (IC
50
, µg mL
−1
).
Antioxidant peptides from chia expeller generated by sequential
hydrolysis (H-A90-F90) showed antioxidant activity with TE values of
4.87 ±0.13 mmol L
-1
mg
−1
(IC
50
=56.23 ±1.07 µg mL
−1
) and of 1.55 ±
0.02 mmol L
-1
mg
−1
(IC
50
=89.12 ±1.04 µg mL
−1
) for ABTS and DPPH
assays respectively, demonstrating an antioxidant capacity higher than
that observed for the unhydrolyzed sample. These TE and IC
50
values
obtained from chia expeller were similar to those reported by other re-
searchers for hydrolysates obtained sequentially with Alcalase and Fla-
vourzyme using chia seeds as starting material (Segura-Campos et al.,
2013), checking again that also the expeller of chia seeds turns out to be
an excellent substrate for obtaining protein hydrolysates with
outstanding antioxidant activity, regardless of the enzyme system used
(Cotabarren et al., 2019).
ABTS antioxidant activity was also evaluated for hydrolysates ob-
tained from Andean crops under the same conditions (two-stage hy-
drolysis with 90 min-Alcalase and 90-min Flavourzyme). Chirinos et al.
(2020) reported TE values of 1.70 µmol mg
−1
and 1.62 µmol mg
−1
for
hydrolysates obtained from quinoa (Chenopodium quinoa) and kiwicha
(Amaranthus caudatus) respectively. Similar results (1.79 µmol TE mg
−1
)
were found for hydrolysates from Lupinus mutabilis commonly known as
tarwi, Andean lupine or chocho (Chirinos et al., 2021) and for enzyme
assisted hydrolysates from sacha inchi seeds (Plukenetia volubilis) with a
TE value of 1.2 µmol mg
−1
(Chirinos, Pedreschi, & Campos, 2020). On
the other hand, lower values of antioxidant activity with DPPH and
ABTS were reported by Pi˜
nuel et al. (2019) for simulated gastric and
duodenal hydrolysates of white, red, and black Quinoa using an in vitro
model. Therefore, once again, we can highlight that the chia expeller is
an excellent source of antioxidant BAPs, as rich as the whole seed.
3.2.2. ACE-inhibitory activity
Pharmacological therapies to control arterial hypertension consist of
using ACE inhibitors, such as captopril, enalapril, alacepril and lisino-
pril, among other alternatives (Ramalingam et al., 2017). However, the
use of antihypertensive drugs can produce side effects, such as head-
ache, cough, taste changes, skin rashes, among others. That is why the
search for natural products with potential ACE-blocking activity is a
promising alternative for its use in the control of blood pressure (Girgih
et al., 2015; Jemil et al., 2016). ACE inhibitor peptides of different an-
imal and plant proteins have been reported, including soy proteins (Gu
& Wu, 2013) and common bean proteins (Mojica et al., 2017).
In this work, the ACE inhibitory activity of chia expeller hydrolysate
was analysed. An ACE inhibition of 45% was found for the hydrolysate
H-A90-F90 (35 µg mL
−1
), while the initial protein isolates at the same
concentration (C, 35 µg mL
−1
) showed a 25% of inhibitory activity.
These results were lower than those reported by San Pablo-Osorio et al.
(2019) for the hydrolysates produced from chia seeds (77% ACE inhi-
bition) with Alcalase, trypsin and chymotrypsin, but higher than those
reported for hydrolyzed proteins of amaranth (albumin 1 and globulin,
5% −40% inhibition) and for quinoa hydrolysate (between 20 and 40%
inhibition) with Alcalase (Aluko & Monu, 2003). Differences between
the inhibition produced by the hydrolysates and the control (20% higher
for the hydrolysates) could be due to the generation of hydrolysates with
a mixture of peptides of varying molecular weight, containing peptides
with a molecular weight higher than 10 kDa and, therefore, lower ACE
inhibition activity, together with peptides with a molecular weight
lower than 1 kDa, to which this ACE inhibitory activity is attributed
(Segura Campos et al., 2013). Thus, this work represents the rst report
of ACE inhibitory activity produced from chia expeller hydrolysates,
obtaining results consistent with those reported in seed proteins with the
same sequential hydrolysis system by Segura Campos et al. (2013).
3.2.3. Anticoagulant activity
In recent years, food-derived antithrombotic peptides have attracted
increasing attention as possible health-promoting ingredients aimed at
preventing the formation of thrombi, due to their high biological ac-
tivity, low toxicity, and ease of metabolism in the human body. As we
know, large peptides cannot be adequately absorbed by the gastroin-
testinal system and possible allergic response upon administration
(Coombs & McLaughlan, 1984; Kong et al., 2018). Low molecular
weight peptides are especially effective in terms of reducing such re-
actions or possibilities. Peptides with antithrombotic bioactivity usually
contain 3–20 amino acids residues (Cheng et al., 2019; Wang et al.,
2017).
PT and aPTT are the common blood tests to assess the risk of bleeding
and thrombosis. In this trial, the PT and aPTT of the hydrolysate ob-
tained by sequential hydrolysis H-A90-F90 were determined (Fig. 4).
As can be seen in Fig. 4, antithrombotic peptides from chia expeller
generated by sequential hydrolysis showed an increased in the aPTT and
PT times for serum clotting in a dose-dependent manner (which is an
unexpected result since peptides generally only show anticoagulant
activity for the intrinsic pathway).
Furthermore, the hydrolysates showed a more potent effect than the
control since approximately half of the concentration was required to
produce the same delay in clotting time; thus, 500 µg mL
−1
of control (C)
produced a clotting time delay on the intrinsic pathway from 33.2 ±7.8
s to 58.1 ±0.5 s, while the same concentration of H-A90-F90 produced a
2-fold higher clotting time delay (from 33.2 ±7.8 s to 101.7 ±11.1 s).
Concentrations higher than 600 µg mL
−1
of the hydrolysate H-A90-F90
completely inhibited the aPTT test while a concentration of 2 mg mL
−1
of heparin, the commonly used anticoagulant drug, is required to pro-
duce the same effect (Indumathi & Mehta, 2016). Future studies will be
necessary to evaluate the effect of these peptides on the clotting factors
of these pathway, such as factor VIII, IX, XI, XII and Prekallikrein.
When evaluating the effect of the hydrolysate H-A90-F90 on the
extrinsic coagulation pathway, higher concentrations of hydrolysate
were required to produce a considerable effect on the clotting time, since
800 µg mL
−1
of H-A90-F90 were required to produce a 3-fold increase in
clotting time (from 16.5 ±0.9 s to 47.5 ±0.7 s), while 1500 µg mL
−1
of
the control to produce the same effect, possibly affecting the activity of
vitamin K-dependent factors (II, V, VII and X).
These results are promising since to date, no antithrombotic peptides
from Salvia hispanica have been reported, this being the rst report that
shows that chia expeller hydrolysates present antithrombotic activity.
4. Conclusions
As the world needs healthy foods that respond to increased life ex-
pectancy by reducing the risks of chronic diseases, in this work we re-
ported the in vitro potential antioxidant, antihypertensive and
anticoagulant activities of hydrolysates from chia expeller produced by
sequential hydrolysis with Alcalase followed by Flavourzyme. We could
obtained an extensive hydrolysate (H-A90-F90) with a degree of hy-
drolysis higher than 50%, which favoured the production of low mo-
lecular weight peptides with a decrease in complex proteins as shown by
SDS-PAGE. Besides, H-A90-F90 showed high antioxidant capacity as
well as a moderate increase in the ACE inhibition compared to the
control and, also antithrombotic activity by both coagulation pathways,
highlighting that these extensive hydrolysates from chia expeller present
this unusual dual inhibitory activity for both the intrinsic and extrinsic
coagulation pathways, being suitable for use in nutraceutical formula-
tions for populations at risk of circulatory diseases and comorbidities.
Future research will make it possible to explore extraction methods
at industrial scale to understand the technological viability and eco-
nomic sustainability of these by-products, of interest for food use due to
the added value that these potential antioxidant, antihypertensive and
antithrombotic activities confer on them, contributing to the recircula-
tion of the chia expeller, a waste from the oil industry.
B. Oz´
on et al.

Food Chemistry 380 (2022) 132185
7
Declaration of Competing Interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
Acknowledgements
This study is part of the DSc degree thesis of Brenda Oz´
on, carried out
at the UNLP. B. Oz´
on is a doctoral fellow from the National Agency of
Scientic and Technological Promotion (ANPCyT - Argentina); J.
Cotabarren is a posdoctoral fellow from the Argentine Council of Sci-
entic and Technical Research (CONICET); W.D. Obreg´
on is a member
of the Researcher Career Program of the CONICET and M.G. Parisi is an
established researcher of UNLu.
This work was funded by Departamento de Ciencias B´
asicas of the
Universidad Nacional de Luj´
an (UNLu), UAV Projects 2017 and 2018-
SPU and Ciencia y Tecnología contra el Hambre – MINCYT (2021-
2023) grants to MGP, PICT-2018-03271 (2019-2021) and UNLP I +D
2020 project Code X-920, grants to WDO.
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