© The Authors, 2026, Published by the Universidad del Zulia*Corresponding author: kh.hadef@univ-adrar.edu.dz
Keywords:
Salvia hispanica L.
Green extraction
Antioxidant activity
Antimicrobial activity
Food safety
Sustainable bioingredients
Antioxidant and antimicrobial activities of sustainable Salvia hispanica L. seed extracts against
gram-positive and gram-negative bacteria
Actividades antioxidantes y antimicrobianas de los extractos sostenibles de semillas de Salvia
hispanica L. frente a bacterias Gram-positivas y Gram-negativas
Atividades antioxidantes e antimicrobianas dos extratos sustentáveis de sementes de Salvia hispanica
L. contra bactérias Gram-positivas e Gram-negativas
Khawla Zahra Hadef *
Amina Attou
Nour el houda Madjidi
Rev. Fac. Agron. (LUZ). 2026, 43(1): e254308
ISSN 2477-9407
DOI: https://doi.org/10.47280/RevFacAgron(LUZ).v43.n1.VIII
Food technology
Associate editor: Dra. Gretty R. Ettiene Rojas
University of Zulia, Faculty of Agronomy
Bolivarian Republic of Venezuela.
University Ahmad DRAIA Adrar, Faculty of Natural and Life
Sciences/ Department of Biological sciences, Laboratory of
Saharan Natural Resources, National road number 6, Adrar
PO box 01000, Algeria.
Received: 19-11-2025
Accepted: 29-12-2025
Published: 18-01-2026
Abstract
Salvia hispanica L. (chia) seeds are rich in bioactive compounds
with antioxidant and antimicrobial properties, making them valuable
natural ingredients for food and agro-industrial applications. Food
contamination by Gram-positive and Gram-negative bacteria
remains a major challenge for food safety, highlighting the need
for sustainable natural preservatives. This study aimed to compare
two green extraction methods for recovering bioactive compounds
from Algerian dark chia seeds and to evaluate the antioxidant and
antimicrobial activities of the resulting extracts against Gram-
positive and Gram-negative bacteria. Aqueous Soxhlet extraction
and ethanolic maceration were applied. Extracts were analyzed
for total phenolics and avonoids, antioxidant activity (DPPH
assay), and antimicrobial activity using the disc diusion method
against nine microbial strains, including Gram-positive and Gram-
negative bacteria and Candida albicans. The aqueous Soxhlet
extract exhibited the highest total phenolic content and strong
antioxidant activity (88 % DPPH inhibition at 400 μg.mL
-1
). The
ethanolic maceration extract was richer in avonoids and showed
moderate, dose-dependent antimicrobial activity. Gram-positive
bacteria (Staphylococcus aureus and Listeria monocytogenes) were
most sensitive (up to 12 ± 1 mm), while Gram-negative bacteria
were generally more resistant, except Klebsiella pneumoniae (15 ±
1 mm at 15 mg.mL
-1
). Candida albicans was highly susceptible (14
± 1 mm). Green-extracted chia seed extracts demonstrate signicant
antioxidant and moderate antimicrobial activities, supporting their
potential use in food preservation, sustainable product development,
and eco-friendly agro-industrial applications.
This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.
Rev. Fac. Agron. (LUZ). 2026, 43(1): e264308 January-March ISSN 2477-9409.
2-7 |
Resumen
Las semillas de Salvia hispanica L. (chia) son ricas en compuestos
bioactivos con propiedades antioxidantes y antimicrobianas, lo que
las convierte en ingredientes naturales valiosos para aplicaciones
alimentarias y agroindustriales. La contaminación de los alimentos
por bacterias Gram-positivas y Gram-negativas sigue siendo un
desafío importante para la seguridad alimentaria, lo que resalta la
necesidad de conservantes naturales sostenibles. Este estudio tuvo
como objetivo comparar dos métodos de extracción verde para
recuperar compuestos bioactivos de semillas de chia oscuras de
Argelia y evaluar las actividades antioxidante y antimicrobiana de
los extractos resultantes frente a bacterias Gram-positivas y Gram-
negativas. Se aplicaron extracción acuosa Soxhlet y maceración
etanólica. Los extractos se analizaron para determinar fenoles totales
y avonoides, actividad antioxidante (ensayo DPPH) y actividad
antimicrobiana mediante el método de difusión en disco frente a
nueve cepas microbianas, incluidas bacterias Gram-positivas y Gram-
negativas y Candida albicans. El extracto acuoso Soxhlet mostró el
mayor contenido total de fenoles y una fuerte actividad antioxidante
(88 % de inhibición DPPH a 400 μg.mL
-1
). El extracto de maceración
etanólica fue más rico en avonoides y presentó una actividad
antimicrobiana moderada, dependiente de la dosis. Las bacterias
Gram-positivas (Staphylococcus aureus y Listeria monocytogenes)
fueron las más sensibles (hasta 12 ± 1 mm), mientras que las bacterias
Gram-negativas generalmente fueron más resistentes, excepto
Klebsiella pneumoniae (15 ± 1 mm a 15 mg.mL
-1
). Candida albicans
mostró alta susceptibilidad (14 ± 1 mm). Los extractos de semillas
de chia obtenidos mediante métodos verdes demuestran actividades
antioxidantes signicativas y moderadas actividades antimicrobianas,
respaldando su potencial uso en la conservación de alimentos,
desarrollo de productos sostenibles y aplicaciones agroindustriales
ecológicas.
Palabras clave: Salvia hispanica L., extracción verde, actividad
antioxidante, actividad antimicrobiana, seguridad alimentaria,
bioingredientes sostenibles.
Resumo
As sementes de Salvia hispanica L. (chia) são ricas em compostos
bioativos com propriedades antioxidantes e antimicrobianas,
tornando-se ingredientes naturais valiosos para aplicações alimentares
e agroindustriais. A contaminação de alimentos por bactérias Gram-
positivas e Gram-negativas continua sendo um grande desao para
a segurança alimentar, destacando a necessidade de conservantes
naturais sustentáveis. Este estudo teve como objetivo comparar
dois métodos de extração verde para recuperar compostos bioativos
de sementes escuras de chia da Argélia e avaliar as atividades
antioxidante e antimicrobiana dos extratos obtidos frente a bactérias
Gram-positivas e Gram-negativas. Foram aplicadas extração aquosa
Soxhlet e maceração etanólica. Os extratos foram analisados quanto
ao teor total de fenóis e avonoides, atividade antioxidante (ensaio
DPPH) e atividade antimicrobiana pelo método de difusão em
disco contra nove cepas microbianas, incluindo bactérias Gram-
positivas e Gram-negativas e Candida albicans. O extrato aquoso
Soxhlet apresentou o maior teor total de fenóis e forte atividade
antioxidante (88 % de inibição DPPH a 400 μg.mL
-1
). O extrato de
maceração etanólica foi mais rico em avonoides e mostrou atividade
antimicrobiana moderada, dependente da dose. As bactérias Gram-
positivas (Staphylococcus aureus e Listeria monocytogenes) foram as
mais sensíveis (até 12 ± 1 mm), enquanto as Gram-negativas foram
geralmente mais resistentes, exceto Klebsiella pneumoniae (15 ± 1
mm a 15 mg.mL
-1
). Candida albicans apresentou alta suscetibilidade
(14 ± 1 mm). Extratos de sementes de chia obtidos por métodos
verdes demonstram atividades antioxidantes signicativas e atividade
antimicrobiana moderada, apoiando seu potencial uso na conservação
de alimentos, desenvolvimento de produtos sustentáveis e aplicações
agroindustriais ecológicas.
Palavras-chave: Salvia hispanica L., extração verde, atividade
antioxidante, atividade antimicrobiana, segurança alimentar,
bioingredientes sustentáveis.
Introduction
Salvia hispanica L., commonly known as chia, is an emerging
high-value crop with adaptability to diverse climates and potential for
sustainable cultivation. In Algeria, it oers opportunities to diversify
agriculture and develop functional food and agro-industrial products.
Food contamination by Gram-positive and Gram-negative
bacteria remains a major concern for food safety and public health.
Natural bioactive compounds from plants provide a promising
strategy to inhibit microbial growth, reduce chemical preservatives,
and improve food quality.
Chia seeds are rich in omega-3 fatty acids, dietary bers, proteins,
vitamins, minerals, polyphenols, and avonoids (Rahmoune et
al., 2025; Dib et al., 2021), which exhibit strong antioxidant and
antimicrobial activities (Tavera-Hernández et al., 2023; Tavares et al.,
2018). Green extraction methods, such as aqueous Soxhlet extraction
and ethanolic maceration, allow ecient recovery of these bioactive
compounds while minimizing chemical waste and environmental
impact.
This study aims to evaluate the antioxidant and antibacterial
activities of Algerian dark chia seed extracts against Gram-positive
and Gram-negative bacteria, and to compare the eects of two green
extraction methods on the yield and bioactivity of these extracts.
The work highlights the potential of sustainable chia extracts for
enhancing food safety and producing eco-friendly bioingredients for
agro-industrial applications.
Materials and methods
Plant material
Commercial Salvia hispanica L. (black chia) seeds were
rst carefully sorted and cleaned to eliminate foreign particles
and impurities, ensuring sample purity and analytical reliability.
Subsequently, 10 g of seeds were gently ground at room temperature
to enhance the contact surface with the extraction solvent, thereby
improving the eciency of compound recovery (Silva et al., 2022).
Preparation of extracts
Ethanolic extract (Maceration)
Ten grams of black chia seed powder were placed in 100 mL
of ethanol (1:10 w/v) in a 250 mL Erlenmeyer ask and stirred
continuously at room temperature for 24 h. The mixture was ltered
through Whatman paper, concentrated under reduced pressure using
a rotary evaporator (Heidolph Instruments, Germany), and stored at
4 °C until analysis (Motyka et al., 2023).
This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.
Hadef et al. Rev. Fac. Agron. (LUZ). 2026, 43(1): e264308
3-7 |
Results and discussion
Total phenolic and avonoid contents
Table 1 presents the total phenolic content (TPC) and total
avonoid content (TFC) of black chia seed extracts obtained using
ethanolic maceration (EEM) and aqueous Soxhlet extraction (AES).
TPC is expressed as µg gallic acid equivalents per mg of dry extract
(µg GAE.mg
-1
), and TFC as µg quercetin equivalents per mg of dry
extract (µg QE.mg
-1
).
Table 1. Total phenolic and avonoid contents of ethanolic and
aqueous extracts of S. hispanica.
Extracts
Ethanolic maceration
extract
Aqueous Soxhlet
extract
Total Phenolic (μg GAE.mg
-1
) 60.57 ± 4.60 110.67 ± 5.10
Total Flavonoid (μg QE.mg
-1
). 20.66 ± 0.50 3.60 ± 0.30
A marked variation in the biochemical composition of Salvia
hispanica L. seeds was observed depending on the extraction method
and solvent. The aqueous Soxhlet extract (AES) exhibited the highest
phenolic content (110.67 ± 5.10 µg GAE.mg
-1
), whereas the ethanolic
maceration extract (EEM) showed lower phenolics (60.57 ± 4.60 µg
GAE.mg
-1
) but signicantly higher avonoid content (20.66 ± 0.50 µg
QE.mg
-1
) compared to AES (3.60 ± 0.30 µg QE.mg
-1
), indicating that
solvent polarity strongly aects extraction eciency (Knez Hrnčič
et al., 2019; Capitani et al., 2012). The phenolic content of AES is
consistent with previously reported aqueous chia extracts ~105 - 115
µg GAE.mg
-1
(Capitani et al., 2012), while the elevated avonoid
content in EEM conrms ethanol’s eciency in extracting lipophilic
avonoids (Knez Hrnčič et al., 2019).
The extraction approach was designed to maximize recovery
of bioactive compounds while minimizing environmental impact,
in line with sustainable agronomy and green processing principles
(Cravotto et al., 2025; Clarke et al., 2018). Mild pretreatment
preserved sensitive compounds by avoiding harsh reagents and high
energy consumption. Both ethanolic maceration and aqueous Soxhlet
extraction are low-cost, eco-friendly, and suitable for small- and
medium-scale agro-industrial applications. Ethanol, notable for its
renewability, biodegradability, and safety, is particularly eective for
avonoid recovery, while sequential Soxhlet extraction with water
enhances hydrophilic phenolics and reduces solvent waste (Demirkol
et al., 2022; Clarke et al., 2018).
Antioxidant activity
The aqueous Soxhlet extract (AES) demonstrated strong
antioxidant activity, reaching 88 % inhibition at 400 µg.mL
-1
, which
exceeds the values reported for similar chia extracts ~75 % (Silva
et al., 2022) and conrms the eciency of this extraction method
for polar phenolics. Antioxidant potential, assessed using the DPPH
assay, showed dose-dependent radical scavenging for both extracts,
with AES markedly outperforming the ethanolic maceration extract
(EEM, 25 % inhibition) (Figure 1). The high activity of AES can be
attributed to its enrichment in hydrophilic phenolics and mucilages,
which act synergistically as radical scavengers and stabilizers (Silva
et al., 2022; Ullah et al., 2016; Capitani et al., 2012; Sultana et al.,
2009). These results underscore the potential application of AES as a
natural antioxidant to improve oxidative stability and extend the shelf
life of agro-food products.
Aqueous extract (Soxhlet Extraction)
Ten grams of black chia seed powder were sequentially extracted
in a Soxhlet apparatus with 100 mL of hexane (65 °C), chloroform
(60 °C), ethanol (78 °C), and distilled water (100 °C), each for 6 h.
Solvents were removed with a rotary evaporator, and the nal aqueous
extract was stored at 4 °C (Naja et al., 2022).
Microorganisms and culture conditions
The tested microorganisms included four Gram-positive bacteria
(Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC
49452, Listeria monocytogenes ATCC 15313, Bacillus cereus ATCC
11778), four Gram-negative bacteria (Escherichia coli ATCC 25922,
Klebsiella pneumoniae ATCC 700603, Salmonella enteritidis ATCC
2453, Pseudomonas aeruginosa ATCC 27853), and one yeast, Candida
albicans ATCC 10231. Strains were obtained from the Laboratory
of Applied Microbiology to Food, Biomedical and Environmental
Sciences (LAMAABE), Abou Bekr Belkaïd University, Tlemcen,
Algeria. Cultures were grown 18–24 h and adjusted to 0.5 McFarland
standard (≈10⁸ CFU.mL
-1
).
Determination of total phenolic and avonoid contents
Total phenolic content (TPC) was measured using the Folin–
Ciocalteu method (Pérez et al., 1990; Singleton and Rossi, 1965).
Total avonoid content (TFC) was determined via the aluminum
chloride colorimetric assay (Chang et al., 2002).
Antioxidant activity
Antioxidant activity was assessed using the DPPH assay
according to Brand-Williams et al. (1995) with minor modications.
Extract stock solutions were prepared in methanol and diluted to nal
concentrations ranging from 12.5 to 400 µg.mL
-1
. In 96-well plates,
100 µL of each dilution were mixed with 100 µL of 0.1 mM DPPH
solution, incubated for 30 min in the dark, and the absorbance was
measured at 517 nm. The percentage of DPPH radical scavenging
activity was calculated using the following equation:
Antimicrobial activity
Antibacterial and antifungal activities were assessed using the agar
well diusion method. Mueller-Hinton agar plates were inoculated with
standardized microbial suspensions (CA-SFM, 2025), and four 6 mm
wells per plate were lled with 20 µL of either ethanolic or aqueous
extracts at concentrations ranging from 0.5 to 15 mg.mL
-1
. Gentamicin
(10 µg) was used as an positive control, while the corresponding
solvents served as negative controls. Experiments were performed in
triplicate. The chosen concentration range allowed evaluation of the
dose-dependent response of microorganisms and determination of
both minimal and maximal antimicrobial eects, in accordance with
established agar well diusion protocols for natural extracts (Gonzalez
Pastor et al., 2023; Matuschek et al., 2014). Inhibition zones were
classied as non-sensitive (<8 mm), sensitive (9 - 14 mm), very
sensitive (15 - 19 mm), and extremely sensitive (>20 mm).
Experimental design and statistical analysis
The study followed a completely randomized experimental
design comparing two extraction methods (ethanolic and aqueous) at
dierent concentrations for antioxidant and antimicrobial responses.
All experiments were conducted in triplicate, and results are expressed
as mean ± standard deviation (SD). Statistical analysis was performed
using one-way analysis of variance (ANOVA) to evaluate dierences
among extracts and concentrations. Dierences were considered
statistically signicant at p < 0.05. Statistical analyses were carried out
using Microsoft Excel (Microsoft Corp., USA).
% Inhibition =
A control – (A sample – A blank
A control
X 100
This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.
Rev. Fac. Agron. (LUZ). 2026, 43(1): e264308 January-March ISSN 2477-9409.
4-7 |
Figure 1. Antioxidant capacity of chia seed extracts using the
DPPH Assay.
Antimicrobial activity
Chia seed extracts exhibited dose-dependent antimicrobial activity
against nine microbial strains, including Gram-positive and Gram-
negative bacteria, as well as Candida albicans (gure 2; table 2).
Candida albicans also displayed notable susceptibility (14 ± 1 mm),
conrming the antifungal potential of EEM. These eects are consistent
with previous studies showing that ethanolic extracts of chia seeds inhibit
bacterial and fungal growth due to non-polar metabolites, including
methylated avonoids, terpenoids, and unsaturated fatty acids (Ullah et al.,
2016; Segura-Campos et al., 2014; Topçu et al., 2007).
An inverse relationship between antioxidant and antimicrobial
activities was observed, reecting the distinct chemical nature of
the bioactive compounds: polar phenolics are primarily responsible
for antioxidant eects, whereas non-polar metabolites contribute to
antimicrobial action (Kaurinovic and Vastag, 2019; Clarke et al.,
2018). These ndings conrm the concentration-dependent ecacy
of chia extracts, particularly EEM, against Klebsiella pneumoniae,
Staphylococcus aureus, and Candida albicans, supporting their
potential as natural antimicrobial agents, although their activity is
generally lower than that of conventional antibiotics.
Solvent polarity strongly inuenced both chemical composition
and bioactivity: water favored the extraction of polar, antioxidant-rich
fractions, while ethanol eciently recovered lipophilic, antimicrobial
compounds (Rahmoune et al., 2024; Knez Hrnčič et al., 2019; Ullah
et al., 2016; Capitani et al., 2012). Overall, these results highlight
the multifunctionality of S. hispanica seeds as a source of bioactive
compounds for natural preservatives and sustainable agro-industrial
applications, contributing to circular bioeconomy strategies and the
valorization of crops in semi-arid regions such as Algeria.
Conclusion
This study demonstrated that the antimicrobial and antioxidant
ecacy of Salvia hispanica L. seed extracts is strongly inuenced
by the polarity of the extraction solvent and the sustainability of the
extraction process. Aqueous Soxhlet extraction, rich in polar phenolic
compounds, provided extracts with remarkable antioxidant capacity,
while ethanolic maceration yielded lipophilic bioactives with greater
antimicrobial potential, particularly against Candida albicans.
The complementary bioactivities of these extracts underscore the
multifunctionality of chia seeds as a valuable agricultural crop for the
development of natural agro-industrial additives and eco-innovative
formulations. By using renewable, safe, and biodegradable solvents
such as water and ethanol, this work demonstrates sustainable
processing approaches that minimize environmental impact and
enhance the valorization of agricultural resources.
Table 2. Antimicrobial activities (diameters of inhibition zones in mm) of ethanolic (EEM) and aqueous (AES) extracts of S. hispanica
against various microbial strains.
Extracts
Strains
Control Aqueous Soxhlet extract (AES, mg.mL
-1
) Ethanolic maceration extract (EEM, mg.mL
-1
)
GEN 10 µg 0.5 5 10 15 0.5 5 10 15
Staphylococcus aureus 22 ± 1 6 ± 1 7 ± 1 10 ± 1 10 ± 1 6 ± 1 8 ± 1 9 ± 1 12 ± 1
Enterococcus faecalis 26 ± 1 6 ± 1 6 ± 1 6 ± 1 7 ± 1 6 ± 1 7 ± 1 7 ± 1 8 ± 1
Listeria monocytogenes 19 ± 1 6 ± 1 7 ± 1 8 ± 1 8 ± 1 7 ± 1 8 ± 1 9 ± 1 9 ± 1
Bacillus cereus 20 ± 1 6 ± 1 7 ± 1 7 ± 1 8 ± 1 6 ± 1 7 ± 1 7 ± 1 9 ± 1
Escherichia coli 22 ± 1 6 ± 1 6 ± 1 7 ± 1 8 ± 1 7 ± 1 8 ± 1 9 ± 1 10 ± 1
Klebsiella pneumoniae 15 ± 1 6 ± 1 7 ± 1 8 ± 1 14 ± 1 7 ± 1 8 ± 1 11 ± 1 15 ± 1
Salmonella enteritidis 24 ± 1 6 ± 1 7 ± 1 8 ± 1 9 ± 1 7 ± 1 8 ± 1 9 ± 1 10 ± 1
Pseudomonas aeruginosa 18 ± 1 6 ± 1 7 ± 1 7 ± 1 8 ± 1 7 ± 1 8 ± 1 8 ± 1 9 ± 1
Candida albicans 21 ± 1 7 ± 1 8 ± 1 8 ± 1 10 ± 1 8 ± 1 9 ± 1 10 ± 1 14 ± 1
Inhibition zone diameter (mm); Values are expressed as mean ± SD.
Figure 2. Antimicrobial activity of Salvia hispanica L. seed
extracts against (a) Escherichia coli, (b) Pseudomonas
aeruginosa, (c) Klebsiella pneumoniae, (d) Candida
albicans, and (e) Bacillus cereus.
The ethanolic maceration extract (EEM) demonstrated stronger
inhibition, particularly at 10–15 mg.mL-1. Staphylococcus aureus (12 ±
1 mm) and Listeria monocytogenes (9 ± 1 mm) were highly sensitive,
while Klebsiella pneumoniae (15 ± 1 mm) showed inhibition comparable
to gentamicin (10 µg).
This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.
Hadef et al. Rev. Fac. Agron. (LUZ). 2026, 43(1): e264308
5-7 |
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This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.
Hadef et al. Rev. Fac. Agron. (LUZ). 2026, 43(1): e264308
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