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Research Article
Haddad El Rabey*, Rehab F. Almassabi, Ghena M. Mohammed, Nasser H. Abbas, Nadia Bakry,
Abdullah S. Althiyabi, Ibrahim H. Alshubayli, and Ahmed A. Tayel*
Potent antibacterial nanocomposites from okra
mucilage/chitosan/silver nanoparticles for
multidrug-resistant Salmonella Typhimurium
eradication
https://doi.org/10.1515/gps-2023-0225
received November 02, 2023; accepted February 28, 2024
Abstract: The polymeric nanocomposites (NCs), constructed
from okra (Abelmoschus esculentus) fruits mucilage (OM),
silver nanoparticles (AgNPs), and chitosan (Ch), were fabri-
cated as potential candidates to overcome drug-resistant
Salmonella Typhimurium bacteria. AgNPs were directly
mediated by OM, with 4.2 nm mean diameters. The composed
NCs from Ch/OM/AgNPs were innovatively synthesized and
the various ratios of Ch:OM/AgNPs affected the NCs particles’
size and charges. The infrared analysis of employed mate-
rials/NCs validated their interactions and conjugations. The
antibacterial assays of NCs against different resistant S.
Typhimurium strains indicated the efficiency of polymeric
NCs to inhibit bacteria with significant superiority over stan-
dard antibiotics. The NCs that contained equal ratios from Ch
and OM/AgNPs were the best formulation (mean diameter,
47.19 nm and surface charge, +16.9 mV) to exhibit the stron-
gest actions toward S. Typhimurium. The NCs caused severe
deformation, destruction, and lysis in exposed bacteria, as
traced with scanning microscopy. The biosynthesis of AgNPs
using OM and their nanoconjugation with Ch provided effec-
tual natural biopolymers NCs with enhanced expected bio-
safety and efficiency against drug-resistant S.Typhimurium
strains, which supports their potential applications as disin-
fectant, sterilizing, and curative antibacterial agents.
Keywords: antimicrobial, bactericidal, biopolymers, bio-
synthesis, nanoconjugation
1 Introduction
Biopolymers are beneficial polysaccharides attained from
natural sources (e.g., plants, microbes, algae, and marine
sources); they could be supremely applied in numerous
therapeutic/pharmaceutical products due to their renew-
ability, biocompatibility, and availability [1]. The nano-
forms of biopolymers could provide further bioactivities
and potentialities to assist the functionalities of other bio-
molecules [2]. From these biopolymers, plant mucilages
and marine polymers were proposed to investigate their
potential bioactivities.
Mucilages are mainly plant polysaccharides that are
intracellularly formed, with varied molecular structures
[3]; the mucilage polysaccharides could demonstrate some
vital bioactivities (e.g., anti-inflammatory, immune-modula-
tory). Mucilages are also perfect candidates for micro-/nano-
encapsulation of other bioactive molecules, phytochemicals,
and probiotics; their edibility, solubility, stability, and biode-
gradability advocated their practical health applications [4].
Recently, many plant mucilages were applied to develop
nanomaterials, including nanocapsules, nanofibers, and
nanocomposites (NCs) or biosynthesis and encapsulation
of numerous bioactive molecules and nanometals as an
innovative protocol [5–8].
* Corresponding author: Haddad El Rabey, Biochemistry Department,
Faculty of Science, University of Tabuk, 71491 Tabuk, Saudi Arabia;
Genetic Engineering and Biotechnology Research Institute, University of
Sadat City, Sadat, Egypt, e-mail: helrabey@ut.edu.sa
Rehab F. Almassabi, Abdullah S. Althiyabi, Ibrahim H. Alshubayli:
Biochemistry Department, Faculty of Science, University of Tabuk, 71491
Tabuk, Saudi Arabia
Ghena M. Mohammed: Nutrition Department, Faculty of Science
University of Tabuk, 71491 Tabuk, Saudi Arabia
Nasser H. Abbas: Genetic Engineering and Biotechnology Research
Institute, University of Sadat City, Sadat, Egypt
Nadia Bakry: Bone Marrow Transplantation and Cord Blood Unit,
Mansoura University Children Hospital, Mansoura, Egypt; Department of
Biochemistry, Faculty of Medicine, Mansoura University, Mansoura, Egypt
* Corresponding author: Ahmed A. Tayel, Department of Fish
Processing and Biotechnology, Faculty of Aquatic and Fisheries Sciences,
Kafrelsheikh University, Kafrelsheikh 33516, Egypt,
e-mail: ahmed_tayel@fsh.kfs.edu.eg
Green Processing and Synthesis 2024; 13: 20230225
Open Access. © 2024 the author(s), published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License.
Okra fruits/pods (Abelmoschus esculentus) contribute
to public diet/local dishes of many countries because of
vital nutritional constituents (e.g., protein, fiber, minerals);
okra is frequently utilized for thickening and viscous con-
stancy in numerous soups/broth. Okra pods have consider-
able amounts of calcium, iron, and zinc and contain low
amounts of antinutrients with high mineral bioavailability
[9]. Okra mucilage (OM) comprises randomly coiled poly-
saccharides that contain galactose, galacturonic acid, and
rhamnose; the repeated units in the OM structure are (1-4)-
galacturonic acid and (1-2)-rhamnose with some disac-
charide side chains [1,6]. OM, like other biopolymers, has
many advantages compared to synthetic polymers; they
are safe, nonirritant, biocompatible, eco-friendly, biode-
gradable, and chemically inert [10]. OM is daily consumed
in regular diets and therefore guaranteed for biosafety and
compatibility without toxicological studies [11]. The high
viscous and slimy properties of OM could be advantageous
as an encapsulating polymer for formulating sustained-
release medications [1,11]. OM was investigated and demon-
strated as a functional nutritional ingredient with numerous
bioassays; OM possessed remarkable antimicrobial, antiox-
idant, hypoglycemic, antitumor, anti-ulcer, anti-toxication,
and cholesterol-lowering capacities [10–15].
From the most promising marine biopolymers, chit-
osan (Ch), which is a positively charged active biopolymer
derived after chitin deacetylation, comprises linear units of
N-acetyl-D-glucosamine and dissolves in low pH solutions
due to the reactivity of its basic amino groups with acidic
radicals. The cationic attributes of Ch provide remarkable
functionalities such as antimicrobial capabilities (through
interacting/attaching anionic microbial membranes, DNA,
RNA, and enzymes), encapsulation efficiency (via upholding
of further negatively charged biomolecules and nanomater-
ials), biosorption (by adoption of numerous toxic and hazar-
dous molecules), and anticancer potentiality (e.g. interactions
with cancer cells and their interior organelles) [16–18].
Composited organic/inorganic materials from diverse
types that have one phase (at least) in the “nano”scale are
called NCs [19]; NCs have advantageous characteristics
over their parent materials with prominent bioactivities
and functionalities [19,20]. Ch (in its bulk or nano form)
has an extraordinary ability to evolve in bioactive NC con-
structions with other biopolymers, phytocompounds, and
nanometals [8,21–23].
The metallic nanoparticles (i.e., nanometals) were employed
in numerous biomedical and pharmaceutical applications, and
the antimicrobial effectiveness of nanometals was documented,
basically due to cytotoxicity toward microbial pathogens and
interactions with their membranes/interior pathways [24–27].
Mostly, silver nanoparticles (AgNPs) were extensively documented
for their antimicrobial actions [24,25], involving Ag
+
ions release,
ROS (reactive oxygen species) generation in the inner/outer
microbial membranes, interference with the cellular mem-
brane, ribosome-mitochondrial disruption, and nucleic acid
interruptions. The biological synthesis of AgNPs is much
more advantageous compared to physical synthesis and
chemical-based approaches; biosynthesis is biosafe, envir-
onmentally friendly, energy- and cost-saving, which gener-
ates homogenous and bioeffective NPs [25–27].
The encapsulation/conjugation of AgNPs within biopo-
lymer and phytocompound coats can impressively mini-
mize their probable toxicities to human cells/tissues while
promisingly preserving or slightly increasing the bioactiv-
ities toward microbial pathogens and invading cells [28–31].
The global foodborne infections by Salmonella spp.
exceeded 115 million humans annually, resulting from
diverse serovars and strains [32]. Among them, S. Typhi-
murium, “i.e. Salmonella enterica serovar Typhimurium,”
caused most the non-typhoidal salmonellosis in humans
worldwide. The resistances of S. Typhimurium to multiple
antibiotics were reported in different countries, antibio-
tics, and isolates [33].
Accordingly, the aims of the present work were the
extraction of OMs and their innovative usage for AgNP
biosynthesis, the construction of bioactive NCs from Ch,
OM/AgNPs, and to employ them as antibacterial candidates
to suppress S. Typhimurium-resistant strains.
2 Materials and methods
2.1 Materials and reagents
The organically cultivated okra (Abelmoschus esculentus)
fruits, with 24–28 mm length, were obtained from the ARC
“Agricultural Research Center, Giza, Egypt.”All chemicals,
microbiological media, reagents, and dyes used in the current
study (e.g., sodium hydroxide [NaOH]; ethanol [96%]; silver
nitrate [AgNO
3
]; chitosan [≥80% deacetylation degree; ∼100 kDa
molecular weight; CAS 9012-76-4]; sodium tripolyphosphate;
acetic acid glacial; potassium bromide [KBr]; and p-iodoni-
trotetrazolium violet dye) were purchased from Sigma-
Aldrich Co. (St. Louis, MO), unless other source is specified.
2.2 Extraction of OM
OM was extracted from A. esculentus fruits, as adapted
from Freitas et al. [34]. First, the okra fruits were manually
2Haddad El Rabey et al.
sliced to obtain a 2–3 mm height using a stainless steel
cutter, and the resulting pieces were immersed in DIW
(deionized water) at 1:30 w/v ratio at room temperature
(RT, 25 ±2°C). Using 1 M NaOH, the pH value was adjusted
to 10.1 ±0.2. Afterward, this mixture was (122 ×g) thor-
oughly stirred for 45 min at RT (∼25 ±2°C). The OM was
separated from okra fruit residues by pressing through a
double sterile cloth filter. For OM precipitation, an equal
volume of ethanol (96%) was mixed with the former extract,
and their mixture was statically kept at a cold (4 ±1°C) tem-
perature for 125 min. The precipitated OM was detached via
centrifugation (4,650 ×g) and freeze-dried.
2.3 Preparation of OM/AgNPs
The procedure for AgNP reduction/mediation using OM
was modified from a recent protocol [35]. First, fresh solu-
tions of AgNO
3
(10 mM) and OM (0.1%, w/v) were separately
prepared in DIW at RT, using stirring at 230 ×gfor 60 min.
The solutions were passed through a syringe (0.45 µm)
filter, and 25 mL of OM solutions were dropped in equal
amounts of AgNO
3
solution (while stirring at 580 ×g) for
95 min. The color change of the mixture solution into
blackish brown could be visually seen as an indicator of
OM/AgNPs formation. The formed NPs were harvested
from the solution through 35 min centrifugation at 9,800
×g(2-16 KL, Sigma; Osterode am Harz, Germany), washing
with DIW, re-centrifugation, and freeze-drying. To attain
plain AgNPs, the DIW washing and re-centrifugation pro-
cesses were repeated five times to eliminate most of the
attached OM.
2.4 Construction of Cht/OM/AgNPs NCs
The assembly of the NCs from Ch/OM/AgNPs followed these
steps [8]: solutions of 0.1% (w/v) were made from OM/
AgNPs (in DIW) and Ch (in 1.5%, v/v aqueous acetic acid
solution); the solutions were individually sonicated for
14 min. About 200 mg of TPP (Na-tripolyphosphate) was
dissolved in 20 mL of OM/AgNP solution and re-sonicated.
Then, the Om/AgNPs-TPP solution was gently dropped
(∼20 mL·h
−1
) into Ch solution under speedy stirring (735 ×
g)forNCformation:
•F1 (2 OM/AgNPs: 1 Ch)
•F2 (1 OM/AgNPs: 1 Ch)
•F3 (1 OM/AgNPs: 2 Ch).
Stirring was continued for a further 40 min, and then
the formed NCs were harvested via centrifugation (10,720 ×
g), DIW washing, recentrifugation, and freeze-drying. For
plain Ch formation, the solvated TPP of step 4 was directly
dropped without the Om/AgNP amendment.
2.5 Nanomaterial/NC characterization
2.5.1 Fourier transform infrared (FTIR) spectroscopy
analysis
FTIR spectroscopy was utilized for analyzing the functional
groups/biochemical bonds in the materials/NCs employed
in the investigation and their interactions, including Ch, OM,
OM/AgNPs, and Ch/OM/AgNPs. FTIR (JASCO, FT-IR-360, Japan)
spectra were appraised after mixing materials/NC powders
with KBr; their IR spectra (transmission mode) were mea-
sured in the wavenumber range of 4,000–450 cm
−1
.
2.5.2 Nanomaterial size and charge
The constructed nanomaterials/NCs were dissolved in DIW
and sonicated at 43 W for 15 min. Then, the distribution
characteristics of particle sizes (Ps), zeta (ζ) potential, and charges
were estimated using a Malvern™Zetasizer (Worcestershire, UK)
at RT, employing the dynamic light scattering (DLS) .
2.5.3 Scanning (SEM) and transmission (TEM) electron
microscopy
SEM (JEOL IT100, Tokyo, Japan) and TEM (Leica; Leo-0430;
Cambridge, UK) imaging elucidated the nanomaterial/NC
morphology, Ps, and distributions. DIW suspensions of
nanomaterials/NCs (OM/AgNPs, F1, F2, and F3) were sonicated
for inspection. The OM-mediated AgNPs were inspected via
TEM after dropping onto copper grids, vacuum-dehydrating for
32 min, and subjecting to TEM imaging at 20 kV. The NC solutions
were SEM screened after mounting onto carbon discs (self-adhe-
sive), coated with palladium/gold (the coater: E5100 II, Polaron
Inc.,PA),andinspectedat10–15 kV operating acceleration.
2.6 Anti-Salmonella activity
2.6.1 Bacterial strains
Different strains of Salmonella Typhimurium “Salmonella
enterica ssp. enterica serovar Typhimurium”were screened
Antibacterial nanocomposites of okra/chitosan/silver NPs 3
in challenging assays; they included the standard strain
ATCC-700408 (multi-drug-resistant) and the isolates M and
H (isolated from minced beef and worker’shands,respec-
tively). The S. Typhimurium isolates were principally
identified based on Gram stain, catalase/oxidase reaction,
morphology, and endospore formation. Standardized proto-
cols (ISO 6579-1:2017 and ISO/TR 6579-3:2014) were followed
to identify the Salmonella strains [36,37]. The identification
was further continued with (an API automated system) for
the analytical profile index “BioMérieux Vitek-II System,
France”according to the manufacturer’sinstructionsand
further confirmed using 16 S rRNA analysis [38]. The used
isolates exhibited multiple drug resistances to ampicillin,
tetracycline, streptomycin, and sulfonamide. Nutrient agar/
broth (NA and NB; Difco Lab., Detroit, MI, USA) was used for
bacterial activation and propagation and challenged aero-
bically at 37 ±1°C.
2.6.2 Qualitative inhibition zone (IZ) antibacterial assay
The experiments were mostly conducted without direct light
sources (e.g., in the dark) to eliminate the probable effects of
light on NP antimicrobial activity. The S. Typhimurium
strain cells spread on NA media were individually chal-
lenged using paper assay discs (6 mm diameter) that carried
25 µL of nanomaterials/NC solutions (with 10 mg·mL
−1
con-
centration), and discs were positioned onto inoculant sur-
faces. These discs were loaded with Ch, OM/AgNPs, F1, F2,
and F3 NCs, and the challenged plates were incubated for
28–36 h until the IZ development. The mean diameters of IZs
were measured and computed.
2.6.3 Quantitative minimum inhibitory
concentration (MIC)
The MICs of Ch, OM/AgNPs, F1, F2, and F3 NCs against
challenged S. Typhimurium strains were gauged using
the “micro-dilution”protocol [39]. The grown Salmonella
cultures in NB (∼3×10
7
cells·mL
−1
) were challenged by suc-
cessively amended concentrations from nanomaterials/NCs
(within 1–100 µg·mL
−1
range), using (96-well) microplates.
Then, a chromogenic indicator (p-iodonitrotetrazolium violet;
4% w/v) was added to the challenged microplate wells after 20
±2 h of incubation, which provides red formazan color with
cells’viability. For bactericidal action confirmation, 100 µL
from colorless wells that were suspected to have MICs were
plated onto fresh plain NA plates to detect potential growth.
MICs indicate the least nanometal concentrations, which
inhibited exact bacterial development in microplates and
subsequent NA plates.
2.6.4 SEM observation of NC-challenged bacteria
After exposure to Ch/OM/AgNPs (F2 formulation), at rele-
vant MIC values against S. Typhimurium ATCC-700408,
cells were incubated for 3, 6 and 9 h, and the apparent
morphological variations after treatments were photo-
graphed by SEM. The logarithmic-grown S. Typhimurium
cells were amended by the NC in NB. Then, cells were
harvested via centrifugation (4,850 ×g), washed with saline
buffer, dehydrated with ethanol on SEM stubs, gold/palla-
dium coated, and screened for structural distortion/defor-
mation compared to control cells.
2.7 Statistical analysis
SPSS v17.0 package software (SPSS Inc., Chicago, IL, USA)
was used for statistical data analysis. Standard deviations
and averages after triplicate measurements were calculated;
the significances between them were computed using ANOVA
(one-way) and t-tests, with p≤5%.
3 Results and discussion
The aims of the current research were the fabrication of
OM-mediated AgNPs and their conjugation with Ch to gen-
erate bioactive antimicrobial NCs against S. Typhimurium.
The nanomaterials/NCs were synthesized using facile pro-
tocols and characterized to assess their attributes and anti-
bacterial bioactivities.
3.1 FTIR assessment
FTIR analysis of composite materials can highlight their
major biochemical groups/bonds and reveal the effects of
interactions between the components (Figure 1). The pat-
terns of interacted materials (e.g., OM, OM/AgNPs, Ch, OM/
Ch, and Ch/OM/AgNPs) illustrated their basic biochemical
groups and their changes after nano-compositing.
For OM, the existence of many peaks around 3,420 cm
−1
indicated the vibrated stretches of N–HandO–H bonds of
carboxylic acid; the –OH presence in the pattern employs
the hydrophilic appeal of that polymer. The 1,738.5 cm
−1
peak is indicative of C]O stretch (mainly in alacturonic
acid), whereas the region of 1,230–868 cm
−1
could represent
acarbohydratefingerprint [40,41]. The IR spectrum at
4Haddad El Rabey et al.
1,636.8 cm
−1
is attributed to the ionized carboxyl, whereas
the 2,921.6 cm
−1
band is attributed to the –CH
2
groups in
cellulose/hemicelluloses and the 1,738.5 cm
−1
is attributed
to the ester carbonyl of OM [5,42,43].
Additionally, the so-called “fingerprint region,”which is
attributed to polysaccharides, could be detected between
1,400 and 1,000 cm
−1
[44], and these representative polysac-
charides’bands were documented also within 1,200–950 cm
−1
[34]. According to these fingerprints, the 1,254.4 cm
−1
band
was assigned to C–O stretching, whereas the 1,052.1 cm
−1
referred to C–O–C group stretching in complex polysacchar-
ides. Furthermore, the 1,628.2 cm
−1
band could be assigned to
amide I and is located in a highly sensitive region of proteins’
secondary structures and attributed to stretched C]Ovibra-
tions of peptides’linkages [45]. This remark suggested that,
besides polysaccharides, the OM could also comprise some
proteinaceous materials, which is expected as the biopolymer
extraction did not involve the deproteinization step [44]. The
band at 3,273.4 cm
−1
signified the intra- and inter-molecular
H-bonding within associated hydroxyl groups to the carbohy-
drate structure [46].
The reduction and conjugation of AgNPs with OM
resulted in notable alterations in their IR spectra (Figure 1,
OM/Ag) compared to the plain OM spectrum. The spectra
changes included the disappearance of several bands (e.g.,
at 621.3 cm
−1
, within the 1,288.4–1,446.3 cm
−1
range, at 1,623.8,
1,686.5, and 3,620.3 cm
−1
), which specify that the disappeared
groups interacted with AgNP ions and broke their bonds
through biosynthesis/reduction. The changes also included
the emergence/sharpening of numerous peaks in the
OM/AgNP spectra (e.g., at 491.6 and 762.4 cm
−1
,withinthe
894.5–1,119.6 cm
−1
range, 1,788.9–2,863.1 cm
−1
range and at
3,404.6 cm
−1
), which markedly indicate the development of
novel bonds amid OM biomolecules with AgNPs [8,35,47].
The IR spectrum of Ch (Figure 1, Ch) could assign the
key bonds/groups that specify standard chitosan [20,48].
The key characteristic indicators in the plain Ch spectrum
were perceived around 3,425 cm
−1
(stretching vibration of
N–H and O–H groups), 2,924.2 cm
−1
(stretching vibration of
C–H aliphatic groups), 1,657.5 cm
−1
(stretched vibration
of amide II NH bonds), 1,116.4 cm
−1
(–OH vibrational stretching
in the C3 carbon), and 1,033.8 cm
−1
(–OH vibrational stretching
in the C6 carbon) [16,49,50]. Consistently, in the Ch spectrum,
thewidebandaround3,420cm
−1
is the key band for interac-
tion with cross-linker agents and other biomolecules in the Ch-
based NCs [21,48].
The OM/Ch/AgNPs show the NC’s main bonds/groups
(Figure 1), which reflect the consequences of component
interactions. In the NC spectrum, several bands appeared
to be derived from Ch (indicated with red zones), and
others were derived from OM/AgNPs (indicated with green
zones), which could affirm physical and electrostatic con-
jugation with combined components, mostly because of
dissimilar charges of these components [8,51].
3.2 Optical assessments of OM-mediated
AgNPs
The bioreduction/transformation of AgNO
3
to AgNPs, via
OM mediation/stabilization, could be perceived visually
(Figure 2). The color of the OM and AgNO
3
solution mixture
changed from faint yellow to blackish brown within 40 min
of the bioreduction process (Figure 2a).
Figure 1: Infrared assessment of employed materials/composites
including okra mucilage (OM, synthesized AgNPs with mucilage (OM/
AgNPs), chitosan (Ch), and their conjugates.
Antibacterial nanocomposites of okra/chitosan/silver NPs 5
The spectroscopic (UV-vis) analysis of OM-mediated
AgNPs authenticated their reduction/transformation into
nanoform (Figure 2b), which was verified by the lambda-
max (420 nm) of the reduction solution color. The TEM
images of the OM-biosynthesized AgNPs could verify the
formation and dispersion of NPs, with an average diameter
of 4.32 nm (Figure 2(T)).
Optical characterization shows the increased potenti-
ality of OM to generate AgNPs, which is basically attributed
to the surface plasmon resonance (SPR) of biosynthesized
AgNPs that was reported to have a λmax of around 420 nm
[27,52,53]. The UV sharp single peak and the dark blackish
color of the biosynthesis matrix confirmed the efficacy of
OM in reducing, stabilizing, and capturing AgNPs [23,54].
Additionally, the elemental analysis via energy disper-
sive X-ray (EDX) that coupled with TEM showed the predo-
minance of Ag, C, and O elements in the OM-biosynthesized
AgNPs (supplementary materials; Figure S1).
3.3 Structural attributes
The physiognomies of nanomaterial/NC structures (e.g.,
OM/AgNPs and the constructed NCs from Ch:OM/AgNPs)
were screened through DLS examination (Table 1) and elec-
tron microscopy (Figure 3). The parent components (e.g.,
Ch and OM/AgNPs) carried contrasted charges (+37.5 and
–26.3 mV, respectively), and their mean particles greatly
differed (>1,000 and 4.21 nm, respectively), as shown in
Table 1 and Figure 2(T).
The mean sizes and charges of constructed NCs (F1, F2,
and F3) indicated the effect of Ch:OM/AgNP mixing ratios
on the generated NC particles (Table 1 and Figure 3). The
SEM images of NC formulations illustrated consistent sizes
with the measured sizes by DLS analysis; the least average
NC size was achieved in F2 (47.19 nm), followed by F3 and
then F1 composites (54.58 and 78.65 nm, respectively). The
F1 composites had negative surface charges (−18.6 mV), and
both F2 and F3 formulations carried positive charges (+16.9
and +27.3 mV, respectively). The microscopic images empha-
sized the well-distributed and miniature NC particles, espe-
cially in the F2 construction (Figure 3b).
The presented innovative protocol for the production
of Ch/OM/AgNP composites (F1, F2, and F3) succeeded
in generating diminished NC particles, as verified by
Figure 2: Visual look (a), the UV-vis assessment pattern (b) of bio-
synthesized AgNPs with Abelmoschus esculentus mucilage (OM), and
transmission microscopy imaging (T) of OM-synthesized AgNPs.
Table 1: Physiognomies of screened materials/NCs
Nanomaterials Size range (nm) Mean size (nm) Charge (mV)
OM/AgNPs 1.91–16.72 4.21 −26.3
Ch >1,000 >1,000 +37.5
F1 (2 OM/AgNPs: 1 Ch) 33.26–203.18 78.65 −18.6
F2 (1 OM/AgNPs: 1 Ch) 20.12–128.77 47.19 +16.9
F3 (1 OM/AgNPs: 2 Ch) 28.80–195.53 54.58 +27.3
6Haddad El Rabey et al.
preceding examinations. This nanoconjugation was recently
introduced using other plant mucilage (garden cress seeds)
and nanometals (i.e., selenium NPs) [8], which validated this
protocol for effectual synthesis of biopolymer NCs. The key
suggested factor for generating such biopolymer NCs is the
apparent opposite charges carried onto parent components’
surfaces (e.g., positive in Ch and negative in interacting
materials). The interaction and formation of NCs among
oppositely charged biopolymers were stated earlier; they
involved Ch as the positively charged polymer in addition
to other negatively charged types (e.g., alginate, fucoidan,
carrageenan, ulvan, gums, nanometals) [8,22,26,55]. The resul-
tant NCs from these conjugations normally have higher sta-
bility, bioactivity, and bio-functionality [8,26,56].
Herein, the anionic OM/AgNP complex could interact
with the cationic Ch to develop an innovative polyelectro-
lyte complex (PLC); the net surface charge of such PLC
mainly depends on the involved ratios from component
electrolytes [56]. The value of NC charges (Zeta potentiality)
can significantly affect their stability/dispersion inside aqu-
eous suspensions through repulsed electrostatics between
particles, which is of influential importance for NC bioac-
tivities [57].
3.4 Anti-Salmonella Typhimurium activities
The antibacterial potentialities of biomaterials/NCs (OM/AgNPs,
Ch,F1,F2,andF3formulations)againstS. Typhimurium isolates
were proven using qualitative/quantitative assays (Table 2). The
fabricated NCs (F1, F3, and F3) exhibited more forceful antibac-
terial actions compared to their parent components (Ch and
OM/AgNPs). This was manifested by the wider IZ diameters
and lesser MICs. Regarding these measurements, the most
Figure 3: SM images of NCs produced from biosynthesized silver
nanoparticles with okra mucilage:chitosan with proportions of (a) 2:1;
(b) 1:1; and (c) 1:2.
Table 2: Anti-Salmonella Typhimurium activities of biosynthesized silver nanoparticles with okra mucilage (OM/AgNPs), chitosan (Ch), and their
composites against different isolates from drug-resistant strains*
Antimicrobial agent Salmonella Typhimurium strains
S. Typhimurium ATCC S. Typhimurium M S. Typhimurium H
IZ (mm) MIC (µg·mL
−1
) IZ (mm) MIC (µg·mL
−1
) IZ (mm) MIC (µg·mL
−1
)
OM/AgNPs 17.2 ± 0.9
a
35.0 18.7 ± 1.3
a
32.5 15.3 ± 0.8
a
45.0
Ch 11.3 ± 0.6
b
>100.0 11.9 ± 0.9
b
95.0 10.4 ± 0.9
b
>100.0
F1 (2 OM/AgNPs: 1 Ch) 23.8 ± 1.5
c
10.0 25.4 ± 1.4
c
10.0 19.8 ± 1.6
c
15.0
F2 (1 OM/AgNPs: 1 Ch) 27.3 ± 2.3
d
7.5 28.6 ± 2.4
d
7.5 22.9 ± 2.8
d
12.5
F3 (1 OM/AgNPs: 2 Ch) 19.8 ± 1.2
e
12.5 22.2 ± 1.6
e
10.0 17.2 ± 2.2
e
15.0
Chloramphenicol 17.6 ± 0.8
f
15.0 22.1 ± 1.7
e
12.5 18.9 ± 1.5
c
15.0
*
Dissimilar letters within a column (superscripts) designate significance of differences (p≤0.05).
Antibacterial nanocomposites of okra/chitosan/silver NPs 7
effectual NCs were F2 (1 OM/AgNPs: 1 Ch), which exhibited the
highest efficacy toward the entire challenged strains. F2 could
significantly exceed the action of chloramphenicol. The subse-
quent bioactive NCs were F1 and F3, respectively.
The bacterial strain responses varied toward examined
antibacterial NCs; S. Typhimurium M was the most suscep-
tible strain, whereas S. Typhimurium H exhibited the upper-
most resistance among challenged strains (Table 2). The
variation in bacterial resistance pattern to NCs validated
that they belonged to different strains.
The key antibacterial agents in OM were illustrated to
include many antinutrient components (e.g., tannins, phytic
acid, essential oils, and flavonoids), which affect bacterial
development [6]. Additionally, some lipid components of OM
(mainly stearic and palmitic acids) were suggested for the
antimicrobial potentiality of OM-based films [7,58]. Some
investigations appointed that OM-based composites with
other biopolymers (e.g., alginate, starch, carboxymethyl cel-
lulose) have enhanced ability to carry/encapsulate bioactive
molecules (e.g., zinc oxide, oxcarbazepine) and improve
their functionalities (e.g., as antimicrobial agents) [5–7]. Cur-
rent results show agreements with these investigations as
the OM-based NCs with Ch could strengthen the AgNP bio-
cidal activity toward S. Typhimurium strains. Numerous
investigations highlighted the powerful action of AgNPs to
control drug-resistant microorganisms, particularly with AgNPs
embedding within the nanopolymer matrix (e.g., Ch, cellulose,
alginate, and their composites) [25]. The main AgNPs' antibac-
terial actions are attributed to the ions’ability to bind/interact
with thiol groups (R-SH) in bacterial membrane proteins to
obstruct respiration and biofunctions of cell walls [24]. As the
Ch cationic nature facilitates its attachment/attraction with the
negative bacterial membranes, Ch was suggested as an ideal
nanocarrier to deliver antimicrobial agents (e.g., OM/AgNPs
here) to the surface or inside the cells to perform their destruc-
tive actions [17].
The associations between NC surface charges and their
biocidal actions (e.g., bactericidal and anticancerous) were
reported [8,47,59–61]; F2 (positively charged NCs) had the stron-
gest antibacterial activity here, which involves their ability to
attach to the negatively charged bacterial membranes.
3.5 SEM screening of morphological
deformation of NC-exposed bacteria
The results of exposure to the Ch/OM/AgNP composite on
the morphology, structure, and manifestation of S. Typhimurium
ATCC-700408 are presented in Figure 4. For an imaginable expla-
nation of Ch/OM/AgNPs (formulation F2) antibacterial actions,
SEM visualizations were screened against the standard S. Typhi-
murium drug-resistant strain. The 0-time exposed cells exhibited
ordinary, healthy, and uniform structures; no distortion/deforma-
tion signs were observed (Figure 4(T0)). After exposure to Ch/OM/
AgNPs for 3 h (Figure 4(T3)), remarkable deformation/distortion
signs were initiated on bacterial membranes, with observable
attached NCs to cell surfaces. The signs of cells’deformations/
destructions were observable after 6 h exposure to Ch/OM/AgNPs
(Figure 4(T6)); the NC particles covered most of the treated cells.
Figure 4: Scanning images of treated Salmonella Typhimurium cells with
the NC from biosynthesized silver nanoparticles with okra mucilage and
chitosan, illustrating the control (T0) and after treatment for 3, 6, and 9 h.
8Haddad El Rabey et al.
Many irregular/inconstant cell shapes appeared in this stage,
with observable cells residues after their lysis. After 9 h exposure
to Ch/OM/AgNPs, most treated S. Typhimurium cells were lysed/
decomposed; their interior exudates and membrane residues
that conjugated the NC particles were most observable (Figure
4(T9)). The matched SEM observations were recorded formerly
after the exposure of varied bacterial types to Ch-based NCs in
conjugation with other biopolymers, nanometals, and phyto-
chemicals [8,23,26]. The synergism of biocidal activities after
nanoconjugation of Ch with other biomolecules (e.g., the F1, F2,
and F3 formulations here) in comparison with their parent con-
stituents (e.g., Ch, OM, and AgNPs) was earlier attributed to Ch
capabilities (depending on surface positivity) to develop NCs
through encapsulation of further molecules from bioactive nano-
materials; these NC bioactivities involve the attachment into
negative bacterial cells/membranes, interruption of their perme-
ability, and obstructing bacterial biosystems [23,27,48].
The Ch biocidal action was shown from captured SEM
images that elucidated the adherence of Ch/OM/AgNPs onto
bacterial cells and assumingly their penetration inside the
cells to destroy their vital biosystems [21,23,26,55,62].
SEM images formerly showed that Ch-based bioactive
NCs could possess multiple antibacterial mechanisms via
attaching to negatively charged cell walls/membranes, sup-
pressing the membrane synthesis, affecting membranes’
permeability, penetrating the innermost cells, leaking vital
cellular components, suppressing the metabolic pathways/
functions, and inducing the apoptosis-like death in bac-
terial cells [23,55,62].
Besides the surface positivity of (F2) NC that could
facilitate its attachment onto negative bacterial walls/mem-
branes and/or vital components, the NC content of bio-
synthesized nanometals (e.g., AgNPs) was validated to
possess the powerful antibacterial actions that principally
depend on NP cytotoxicity toward targeted cells via inactiva-
tion/interaction with their physiological pathways [29,63,64].
The main limitation of the current protocol was the potential
biotoxicity of AgNPs, which could be overcome by the bio-
synthesis of nanometals using organic matter and their nano-
conjugation with the Ch biopolymer [65–67]. Nearly all the
materials/components used were derived from natural
sources, which warrant the non-toxicity, biocompatibility,
and sustainability of the process and minimize the potential
biotoxicity of AgNPs [66,68,69].
4 Conclusions
This research targeted the generation of bioactive antimi-
crobial NCs against S. Typhimurium through the fabrication
of OM-mediated AgNPs and their conjugation with Ch. The
nanomaterials/NCs were successfully synthesized using facile
protocols and characterized to assess their attributes and anti-
bacterial bioactivities. OM could mediate AgNP reduction/sta-
bility with a mean Ps of 4.21 nm. The biopolymers formulated
NCs of Ch/OM/AgNPs, which were extraordinarily validated
as effective anti-Salmonella Typhimurium nano-medication.
The formulation F2 (contained equal ratios of OM/AgNPs and
Ch),withameansizeof47.19nmandasurfacechargeof
+16.9 mV, were the most effectual antibacterial NCs against S.
Typhimurium strains and could significantly exceed standard
antibiotic actions. The biosynthesis of AgNPs using OM and
their nanoconjugation with Ch provided effectual natural bio-
polymer NCs with enhanced expected biosafety and efficiency
against drug-resistant S. Typhimurium strains, which supports
their potential applications as disinfectant, sterilizing, and cura-
tive antibacterial agents.
Acknowledgment: The authors extend their appreciation
to the Deputyship for Research & Innovation, Ministry of
Education in Saudi Arabia, for funding this research work
through the project number (0100-1443-S).
Funding information: This work was funded by the
Deputyship for Research & Innovation, Ministry of Education
in Saudi Arabia, through the project number (0100-1443-S)
Conflict of interest: The authors state no conflict of interest.
Author contributions: Conceptualization, H.A.E. and A.A.T.;
methodology, G.M.M., R.F.A., and A.A.T.; software, N.H.A.,
and A.S.A.; validation, A.S.A., I.H.A., and R.F.A.; formal ana-
lysis, G.M.M., and A.A.T.; investigation, M.A.A., E.S.A., and
A.A.T.; resources, M.A.A. and H.A.E.; data curation, N.H.A.,
I.H.A., and R.F.A.; writing –original draft preparation, H.A.E,
N.B., and A.A.T.; writing –review and editing, M.A.A. and
A.A.T.; visualization, G.M.M. and N.H.A.; supervision, H.A.E.
and A.A.T.; project administration, I.H.A., N.B. and A.S.A.;
funding acquisition, H.A.E. All authors have read and agreed
to the presented version of the manuscript.
Data availability statement: All data generated or ana-
lyzed during this study are included in this published article.
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