foods

Article
Antibacterial and Antifungal Activities of Cimbopogon
winterianus and Origanum syriacum Extracts and Essential Oils
against Uropathogenic Bacteria and Foodborne Fungal Isolates
Marwa Rammal 1 , Salam Khreiss 1 , Adnan Badran 2 , Malak Mezher 3 , Mikhael Bechelany 4,5, * , Chaden Haidar 1 ,
Mahmoud I. Khalil 3,6 , Elias Baydoun 7 and Mohammad H. El-Dakdouki 8, *

1 Department of Food Sciences and Technology, Faculty of Agronomy, Lebanese University,

Beirut P.O. Box 146404, Lebanon; [email protected] (M.R.); [email protected] (S.K.);
[email protected] (C.H.)
2 Department of Nutrition, University of Petra, Amman P.O Box 961343, Jordan; [email protected]
3 Department of Biological Sciences, Faculty of Science, Beirut Arab University, P.O. Box 11-5020,
Beirut 11072809, Lebanon; [email protected] (M.M.); [email protected] or
[email protected] (M.I.K.)
4 Institut Européen des Membranes (IEM), UMR-5635, Université de Montpellier, École Nationale Supérieure
de Chimie de Montpellier (ENSCM), Centre National de la Recherche Scientifique (CNRS), Place Eugene
Bataillon, 34095 Montpellier, France
5 Functional Materials Group, Gulf University for Science and Technology (GUST),
Mubarak Al-Abdullah 32093, Kuwait
6 Molecular Biology Unit, Department of Zoology, Faculty of Science, Alexandria University,
Alexandria 21568, Egypt
7 Department of Biology, American University of Beirut, P.O. Box 11-0236, Beirut 11072020, Lebanon;
[email protected]
8 Department of Chemistry, Faculty of Science, Beirut Arab University, Riad El Solh, P.O. Box 11-5020,
Beirut 11072809, Lebanon
* Correspondence: [email protected] (M.B.); [email protected] (M.H.E.-D.)
Citation: Rammal, M.; Khreiss, S.;
Badran, A.; Mezher, M.; Bechelany,
M.; Haidar, C.; Khalil, M.I.; Baydoun,
Abstract: This study focused on testing the antibacterial and antifungal activity of Origanum syriacum
E.; El-Dakdouki, M.H. Antibacterial (O. syriacum) and Cimbopogon winterianus (C. winterianus) extracts and their essential oils (EOs). The
and Antifungal Activities of bacteria were isolated from urine samples and identified by a VITEK assay, and the fungi were
Cimbopogon winterianus and Origanum isolated from spoiled food samples and further identified by MALDI-TOF. The susceptibility of
syriacum Extracts and Essential Oils the microbial isolates was assessed by determining the bacteriostatic and bactericidal/fungicidal
against Uropathogenic Bacteria and effects by the minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal con-
Foodborne Fungal Isolates. Foods centration (MBC/MFC) broth microdilution assay and time-kill test. The antibiofilm activities were
2024, 13, 1684. https://doi.org/ assessed by the antibiofilm screening assays. The bacterial isolates included three Gram-negative
10.3390/foods13111684
isolates (Escherichia coli, Klebsiella pneumonia, and Citrobacter freundii) and two Gram-positive isolates
Academic Editor: Robert (Staphylococcus aureus and Streptococcus intermedius). The fungal isolates included Candida albicans and
L. Buchanan Aspergillus niger. The O. syriacum and C. winterianus extracts exhibited bacteriostatic and fungistatic
activities (MIC 1.25–2.5 mg/mL for the bacterial isolates and 2.5–5 mg/mL for the fungal isolates).
Received: 20 April 2024
However, their EOs exhibited bactericidal (MBC 5–20%) and fungicidal (MFC 1.25–10%) activities,
Revised: 21 May 2024
Accepted: 24 May 2024
meaning that the EOs had a better antimicrobial potential than the extracts. The antibiofilm activities
Published: 27 May 2024 of the mentioned extracts and their EOs were relatively weak. The O. syriacum extract inhibited S.
aureus, S. intermedius, and K. pneumonia biofilms at a concentration of 0.3125 mg/mL and C. albicans
and A. niger biofilms at 0.625 mg/mL. No antibiofilm activity was recorded for C. winterianus extract.
In addition, the packaging of grapes with C. winterianus extract preserved them for about 40 days.
Copyright: © 2024 by the authors. The results reflect the significant antimicrobial activity of O. syriacum and C. winterianus extracts and
Licensee MDPI, Basel, Switzerland.
their EOs, thus suggesting their potential in food packaging and preservation.
This article is an open access article
distributed under the terms and
Keywords: O. syriacum; C. winterianus; extracts; essential oils; antibacterial activity; antifungal
conditions of the Creative Commons
activity; preservation; food packaging
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

Foods 2024, 13, 1684. https://doi.org/10.3390/foods13111684 https://www.mdpi.com/journal/foods

Foods 2024, 13, 1684 2 of 15

1. Introduction
Food packaging faces significant economic, environmental, and public health chal-
lenges. It is time to mobilize all stakeholders to advance materials and technologies toward
solutions that are more friendly to the environment and respectful to the consumer [1].
Usually, food packages are made of plastic. They are essential for food because they provide
protection against the negative influences of the outer environment [2]. For this reason,
consumers have started looking for alternative food packaging materials due to the con-
sumer demand for non-synthetic products and the growing concerns about plastic’s effects
on the environment [2,3].
Plastic can be replaced with biodegradable materials like biopolymers, bioplastics, bio-
nanocomposites, and edible coatings [3]. Healthy alternatives to traditional food packaging
are edible films and coatings. This is why research has shed light on forming biopolymer-
based biodegradable packaging materials [4]. Aliphatic polysaccharides, proteins, and
polyesters are the main biopolymers applied in food preservation because they increase
the shelf life of products. These packaging materials act as barriers which control gas,
humidity, aromas, and lipids exchange with the outside environment [2,5]. They possess
antimicrobial activities, thus protecting the food products against the external environment
and preventing the loss of flavors, texture, and other desirable elements [2,5–7].
Natural polymers from plants or animals have been used by several researchers [2].
Biopolymers are activated by additives including antimicrobials, antioxidants, nutrients,
essential oils (EOs), phenolic compounds, and plant extracts. This improves the food’s shelf
life. For example, adding glycerol and sorbitol to biopolymers can modify their brittleness,
transformability, starch chain, mobility, and moisture absorption [3].
Grape fruit is non-climacteric and perishable after harvest. It begins to deteriorate due
to the loss of water, fungal rot, and oxidation. This causes rachis blackening and turgor
loss, thus affecting the characteristics of the grape product and making it unsaleable [8,9].
Edible coatings (EDCs) have been widely used as preservatives for fresh fruits because they
extend their post-harvest shelf life [8,9]. EDCs form thin layers of macromolecules that are
applied to food surfaces, where they act as semi-permeable barriers to water vapor and
gases. This reduces the respiration of fruits and in turn the loss of weight [8,10]. In addition,
EDCs maintain fruit firmness and give shine to the coated product [8,11]. They can be made
from various polysaccharides. For example, starch is a natural polysaccharide which has a
high molecular weight and is inexpensive, poorly degradable, and well biocompatible [12].
EOs are hydrophobic liquids made up of volatile aromatic compounds that have a
variety of effects associated with their antimicrobial, anti-inflammatory, antioxidant, and an-
ticancer effects like the EO of O. syriacum [13]. At the same time, the EO of C. winterianus has
several therapeutic applications. It is used as a mosquito repellent and as an antiparasitic,
nematocidal, antifungal, and antibacterial agent [12]. The antimicrobial effect of extracts
and EOs is usually caused by phenolics and monoterpenes, especially carvacrol and thymol.
Carvacrol is considered one of the most effective antimicrobials [14]. The effectiveness
of O. syriacum extract against a wide spectrum of pathogenic microorganisms has been
reported [14,15]. In general, the effectiveness is linked to the high carvacrol content in the
extract. The high lipophilic content of O. syriacum EO was linked to significant antibacterial
activity against Gram-negative bacteria, while the high thymol content was more potent
against Gram-positive bacteria. Both thymol and carvacrol are involved in the disruption
of the bacterial cell membrane and the inhibition of ATPase activity [15]. In addition, the
Lebanese Za’atar volatile EO showed inhibitory effects on fungi, especially Penicillium,
Aspergillus, and Fusarium. It acts by inhibiting mycelial growth [16].
The roots and leaves of C. winterianus EOs are shown to have variable antimicrobial
activities. The EO extracted from C. winterianus leaves exhibited potent activity against
Pseudomonas aeruginosa, Streptococcus pyogenes, Staphylococcus aureus, and Staphylococcus
epidermidis. This effect could be due to the individual components present in the EO and
the synergistic mechanism between the EO components. Furthermore, the EOs extracted
from C. winterianus leaves and roots exhibited potent antifungal activity against Aspergillus
Foods 2024, 13, 1684 3 of 15

fumigatus, Aspergillus niger, Trichophyton mentagrophytes, Microsporumcanis, Candida albicans,

and Trichophyton mentagrophytes. This effect could be due to the synergistic effect of the
components of the EO [17].
At the end of the last century, research shifted from biodegradable film to fully degrad-
able biofilms [18]. Various groups of organic antimicrobial agents are used in the packaging
industry, such as antimicrobial peptides, organic acids, enzymes, and agents of natural
plant origin. The function of biodegradable packaging film is the same as that of con-
ventional packaging, which is to protect the food quality and increase its value. To date,
molecules used to assemble fully biodegradable films include polysaccharides, such as
starch [18]. Coatings can be applied to foods by various methods, including spraying,
dipping, spreading, and thin-film hydration [3].
Few studies have explored the antimicrobial activity of Oregano and Cymbopogon. In
this regard, the present work aims to study the antibacterial, antifungal, and antibiofilm
activities of the combined O. syriacum and C. winterianus extracts and EOs against five
uropathogenic bacteria (Escherichia coli, Klebsiella pneumonia, Citrobacter freundii, Staphylococ-
cus aureus, and Streptococcus intermedius) and two foodborne fungi (Candida albicans and
Aspergillus niger). In addition, it aims to develop a biodegradable film from biopolymers,
starch and glycerol, and bioactive substances extracted from C. winterianus in order to
preserve grapes after harvesting.

2. Materials and Methods

2.1. Isolation and Identification of the Bacterial and Fungal Isolates
Five uropathogenic bacteria were used to test the antibacterial potential of the aqueous
and ethanolic O. syriacum and C. winterianus extracts, as well as their EOs. The bacterial
isolates included three Gram-negative bacteria (Escherichia coli, Klebsiella pneumonia, and
Citrobacter freundii) and two Gram-positive bacteria (Staphylococcus aureus and Streptococcus
intermedius). All bacteria were isolated from urine. The isolation was performed by
spreading 100 µL of the urine samples on various selective media. Following that, Gram
staining was applied to separate the isolates into Gram-positive and Gram-negative isolates.
The bacteria were further identified by VITEK (VITEK System 8.02 Version; Shenzhen,
China). The VITEK assay relies on preparing a bacterial suspension in sodium chloride
(NaCl; DB09153, Matangi, Honduras), adjusting it to 0.5 McFarland, and inserting it into
VITEK cards for biochemical tests. The levels of identification are classified as excellent
(96–99%), very good (93–95%), good (89–92%), and acceptable (85–88%). As for the fungal
isolates, two fungi (Candida albicans and Aspergillus niger) were used. These fungi were
isolated from food samples (lemon and jam) by spreading 100 µL of the samples on a
specific medium (Potato Dextrose agar (PDA); HiMedia Laboratories Pvt. Ltd., Thane,
India) and further identified by matrix-assisted laser desorption ionization–time of flight
mass spectrometry (MALDI-TOF) (Autof MS1000 MALDI-TOF MS; Senegal, Africa) [19].
All bacteria and fungi were obtained and isolated in the Advanced Microbiology laboratory
at Beirut Arab University.

2.2. Preparation of the Aqueous and Ethanolic O. syriacum and C. winterianus Extracts and EOs
The aqueous and ethanolic powdered O. syriacum and C. winterianus extracts were
dissolved in each of their solvents (water and ethanol, respectively) to prepare solutions
ranging in concentration between 0.3125 and 5 mg/mL. The EOs were dissolved in 1%
dimethyl sulfoxide (DMSO; Inc. P.O. Box 439, Ghent, Belgium, KY 410455,1-800/367-6935) to
prepare solutions of concentrations ranging between 1.25 and 20% (v:v) [19]. The aqueous O.
syriacum and C. winterianus extracts were combined by mixing equal masses of the powdered
extracts in distilled water to form concentrations ranging between 0.3125 and 5 mg/mL.
The same preparation was performed for the ethanolic extracts. The EOs were prepared
according to Rammal et al. [20] by hydro-distillation. Briefly, the leaves were heated with
distilled water and the vapor was condensed to separate the oils from water. The EOs were
stored at 4 ◦ C. The O. syriacum and C. winterianus EOs were combined by mixing equal
Foods 2024, 13, 1684 4 of 15

volumes of the EOs and dissolving them in DMSO to prepare solutions of concentrations
ranging between 1.25 and 20%. In addition, the chemical composition of the EOs was tested
by Gas Chromatography (GC; Biobase, BK-GC7820, Wolfenbuttel, Germany) as described
by Rammal et al. [20] using an Agilent 6890N network gas chromatograph equipped with
an Agilent 19091S-433HP-5MS column, with dimensions of 30 m × 0.25 mm × 0.25 µm.
Briefly, the oven temperature was programmed to increase from 65 ◦ C to 450 ◦ C at a rate of
3 ◦ C per min. Subsequently, detector scanning was conducted for a duration of 45 min. The
identification of the reported individual components was achieved through a comparison of
the mass spectra and by referring to the GC-MS library.

2.3. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal/Fungicidal

(MBC/MFC) of the Aqueous and Ethanolic O. syriacum and C. winterianus Extracts and EOs by
the Microdilution Assay
The MICs and MBCs of the aqueous and ethanolic O. syriacum and C. winterianus
extracts and EOs were determined against all bacterial and fungal isolates by employing
the micro-well dilution assay. The bacterial isolates were implanted into Muller Hinton
broth (MHB; OXOID Ltd., Basingstoke, UK) until the turbidity matched the 0.5 McFarland
scale. The fungal isolates were implanted into Potato Dextrose broth (PDB, HiMedia
Laboratories Pvt. Ltd., Thane, India) (optical density = 0.08–0.1 at 600 nm). The test was
carried out in sterile 96-well microplates by adding 90 µL of MHB or PDB and 10 µL of
the bacterial and fungal suspensions into each well. Then, 100 µL of the prepared extracts
(ranging between 0.3125 and 5 mg/mL) and their EOs (ranging between 1.25 and 20%)
were added to the wells. Doxycycline (Dox; 100 mg, Actavis, Barnstaple, EX32 8NS, UK)
was used as a reference antibiotic against the bacterial isolates, and cultures of bacteria
and fungi without any treatment were used as negative controls. The plates containing
bacteria were incubated for 24 h at 37 ◦ C, and the plates containing fungi were incubated
for 5 days at 30 ◦ C. Optical density (O.D.) was measured at 595 nm (ELISA microtiter
plate reader, Thermo Fisher Scientific, Shanghai, China), and the MIC was established
as the lowest concentration of extract or EO that inhibited visible growth of bacteria and
fungi. The MBCs/MFCs were determined by inoculating a loop-full of the clear wells into
Muller Hinton agar (MHA; HiMedia Laboratories, Thane, India) plates and incubating
them at 37 ◦ C for 24 h (bacteria) and 30 ◦ C for 5 days (fungi). The lowest concentration
not exhibiting bacterial or fungal growth was recorded as the MBC/MFC. All experiments
were performed in triplicate [19].

2.4. Determination of the Time Needed by the Aqueous and Ethanolic O. syriacum and C.
winterianus Extracts and EOs to Inhibit Bacterial and Fungal Growth by the Time-Kill Assay
The time-kill assay was used to test the time needed by the aqueous and ethanolic
O. syriacum and C. winterianus extracts and their EOs to exert their inhibitory activity against
the bacterial and fungal isolates. The test was performed in 96-well microplates by adding
90 µL of MHB and 10 µL of bacterial suspensions adjusted to 0.5 McFarland and 90 µL of
PDB and 10 µL of fungal suspensions (O.D. = 0.08–0.1 at 600 nm). Then, 100 µL of the MICs
of the aqueous and ethanolic O. syriacum and C. winterianus extracts, as well as their EOs,
were added into the 96-well microplates. The plates were incubated at 37 ◦ C for bacterial
isolates and 30 ◦ C for fungal isolates, and the O.D. of bacterial growth was measured at
595 nm within the time interval (0–24 h), and that of fungal growth was measured within
the time interval (1–5 days). The experiment was repeated at least three times [21,22].

2.5. Detection of the Anti-Biofilm Activity of the Aqueous and Ethanolic O. syriacum and C.
winterianus Extracts and EOs by the Anti-Biofilm Assay
In order to detect the potential of the aqueous and ethanolic O. syriacum and C. winte-
rianus extracts and EOs to prevent biofilm formation, the anti-biofilm formation test was
applied. Bacteria and fungi were incubated in 96-well microtiter plates by adding 10 µL
of standard bacterial or fungal suspension and 90 µL of MHB or PDB into each well. The
plates were incubated at 37 ◦ C (bacteria) and at 30 ◦ C (fungi) for 30 min. After that, the
Foods 2024, 13, 1684 5 of 15

aqueous and ethanolic O. syriacum and C. winterianus extracts and their EOs were added to
the wells at different concentrations (extracts ranging between 0.3125 and 5 mg/mL and
oils between 1.25 and 20%). Dox served as a reference antibiotic (positive control), and
a bacterial culture without any treatment served as a negative control. The plates were
incubated at 37 ◦ C (bacteria) for 24 h and at 30 ◦ C (fungi) for 5 days prior to being washed
five times with sterile distilled water and oven dried (15 min at 60 ◦ C). Then, 100 µL of 1%
crystal violet (CV; 100 g, SURECHEM PRODUCTS Ltd., C8062; Paris, France) was added
to stain the wells. The plates were incubated for 15 min at room temperature. The plates
were then rinsed five times with sterile distilled water to remove the unabsorbed stain. The
biofilms were noticeable as purple rings on the sides of the wells. Then, 100 µL of 95%
ethanol (Dyadic international, Inc. OTCQX: DYAI, Jupiter, FL, USA) was added to the wells
to destain them. An ELISA reader was used to detect the absorbance at 595 nm. The %
inhibition of biofilm formation was calculated using the following equation:

O.D.(negativecontrol) − O.D.(treatedsample)
% Inhibition = × 100
O.D.(negativecontrol)

For the biofilm destruction, a similar experiment was performed but with the incuba-
tion of the bacteria in MHB for 24 h and the fungi in PDB for 5 days to form the biofilms
before treatment. The antibiofilm activity was also determined using the same mentioned
protocol. All experiments were repeated at least three times [19].

2.6. Preparation of the Film

An envelope was prepared to preserve the table grapes. Distilled water was boiled
and then cooled to room temperature. Following that, 30 g/L of starch and 10% glycerol
were added. The solution was heated until the starch gelatinized, and it was allowed to sit
until it cooled to room temperature. Finally, the aqueous extract of C. winterianus (5 mg/mL
and 10 mg/mL) was added to the prepared solution. The grape bunches were washed with
sterile distilled water and immersed in the prepared solutions for 2 min. The samples were
stored at 4 ◦ C after the package was dried. The results of preservation of the grapes with
the extract were monitored after 1, 17, and 37 days of incubation [23].

2.7. Statistical Analysis

The statistical analysis and graphs were carried out and drawn, respectively, in Excel
(Microsoft office 2016, McIntosh, WA, USA). The statistical significance was determined by
t-tests. A p-value < 0.05 was considered statistically significant.

3. Results
3.1. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal
Concentration/Minimum Fungicidal Concentration (MBC/MFC) of the Aqueous and Ethanolic O.
syriacum and C. winterianus Extracts and EOs
The MIC and MBC microdilution assay was performed to detect the bacteriostatic and
bactericidal activities of the aqueous and ethanolic O. syriacum and C. winterianus extracts
and their EOs. The aqueous C. winterianus extract exhibited a bactericidal effect against
K. pneumonia, S. aureus, and S. intermedius (MBCs ranging between 2.5 and 5 mg/mL)
and a bacteriostatic effect against E. coli and C. freundii (MICs ranging between 2.5 and
5 mg/mL). The aqueous O. syriacum extract had a bacteriostatic effect against all bacterial
isolates (MICs ranging between 1.25 and 2.5 mg/mL). The ethanolic C. winterianus extract
exhibited a bactericidal effect against C. freundii and S. intermedius (MBCs ranging between
2.5 and 5 mg/mL) and a bacteriostatic effect against E. coli, K. pneumonia, and S. aureus
(MICs ranging between 0.625 and 2.5 mg/mL). Both extracts were able to inhibit bacterial
growth at relatively low concentrations. However, better antibacterial activity was recorded
for the combined extracts. The aqueous combined extracts exerted bactericidal activity
against C. freundii and S. aureus (MBCs = 5 mg/mL) and a bacteriostatic effect against
E. coli, K. pneumonia, and S. intermedius (MICs = 2.5 mg/mL). The ethanolic combined
Foods 2024, 13, 1684 6 of 15

extract showed bacteriostatic activity against all bacterial isolates (MIC ranging between
1.25 and 5 mg/mL). Regarding the EOs of O. syriacum and C. winterianus, they showed
significant inhibitory action against the bacterial isolates, in which they showed bactericidal
potentials against most of the isolates at relatively low concentrations. The O. syriacum
EO had a bactericidal effect against C. freundii and S. aureus (MBCs ranging between 1.25
and 20%) and bacteriostatic activity against E. coli, K. pneumonia, and S. intermedius (MICs
ranging between 1.25 and 2.5 mg/mL). The C. winterianus EO had a bactericidal effect
against E. coli, C. freundii, and S. aureus (MBCs = 20 mg/mL), and a bacteriostatic effect
against K. pneumonia and S. intermedius (MICs = 2.5 mg/mL). In addition, the combined
EOs exerted stronger bactericidal action against all tested isolates (MBCs ranging between
5 and 20%), except E. coli. For the fungal isolates, C. albicans was more resistant than
A. niger. Both aqueous and ethanolic extracts had fungistatic activity with a better effect
for the ethanolic extracts. The aqueous O. syriacum, C. winterianus, and combined extracts
showed inhibitory action against A. niger only (MIC = 5 mg/mL). However, the ethanolic
extracts had a fungistatic effect against both C. albicans and A. niger (MICs ranging between
2.5 and 5 mg/mL). On the other hand, the EOs had significant inhibitory activity with
fungistatic action against C. albicans (MICs ranging between 1.25 and 5%) and fungicidal
activity against A. niger (MBCs = 10%). The MIC and MBC/MFC results are presented in
Table 1 and Figure 1, Figures S1 and S2. Table 2 shows a comparison between the MICs
of the extracts and EOs used in this study and the MICs of similar extracts and EOs used
on the same bacterial and fungal species from previous studies. In addition, all data were
significant with p-values < 0.05, as presented in Table S1.

Table 1. MICs and MBCS/MFCs of the aqueous and ethanolic O. syriacum and C. winterianus extracts
(mg/mL) and their EOs (%) against the bacterial and fungal isolates.

Bacterial Isolates
MICs and Fungal Isolates
Plant Gram-Negative Bacteria Gram-Positive Bacteria
MBCs/MFCs of the
Extracts and
Extracts (mg/mL) K. C. S. inter- C.
EOs E. coli S. aureus A. niger
and the Oils (%) pneumonia freundii medius albicans

C. winterianus MIC 5 2.5 2.5 2.5 2.5 - 5

AE MBC/MFC - 5 - 5 5 - -
MIC 2.5 2.5 1.25 1.25 1.25 - 5
O. syriacum AE
MBC/MFC - - - - - - -
Combination MIC 2.5 2.5 1.25 2.5 2.5 - 5
of AE MBC/MFC - - 5 5 - - -

C. winterianus MIC 2.5 1.25 2.5 0.625 1.25 5 5

EE MBC/MFC - - 5 - 2.5 - -
MIC - 2.5 1.25 1.25 2.5 5 5
O. syriacum EE
MBC/MFC - - - - - - -

Combination MIC 2.5 1.25 2.5 5 5 2.5 5

of EE MBC/MFC - - - - - - -

C. winterianus MIC 2.5 2.5 5 2.5 2.5 1.25 1.25

EO MBC/MFC 20 - 20 20 - - 10
MIC 2.5 1.25 1.25 - 1.25 2.5 5
O. syriacum EO
MBC/MFC - - 20 1.25 - - 10

Combination MIC 20 5 5 5 5 5 5
of EOs MBC/MFC - 20 20 20 20 - 10
EOs: essential oils, AE: Aqueous extract, EE: ethanolic extract, MIC: minimum inhibitory concentration, MBC:
minimum bactericidal concentration, MFC: minimum fungicidal concentration, “-”: not determined.
 C. khasianus EO 20 V 20 µg/mL 30 µg/mL - 100 µg/mL [27]
C. winterianus EO 0.98 µg/mL - 0.98 µg/mL - - [28]
C. winterianus EO 6 µg/mL - 2 µg/mL 2.5 µg/mL 1 µg/mL [29]
O. syriacum Combined AEs 2.5 mg/mL 2.5 mg/mL 2.5 mg/mL 5 mg/mL -
Current
and Combined EEs 2.5 mg/mL 1.25 mg/mL 5 mg/mL 5 mg/mL 2.5 mg/mL
study
Foods 2024, 13, 1684 C. winterianus Combined EOs 20% 5% 5% 5% 5% 7 of 15
MIC: minimum inhibitory concentration, EOs: essential oils, AE: Aqueous extract, EE: ethanolic
extract, “-“: not determined.

Figure 1. MBC/MFC results of the aqueous and ethanolic O. syriacum and C. winterianus extracts,
and their EOs against the bacterial and fungal isolates (MBC: minimum bactericidal concentration,
EOs: essential oils).

Table 2. Comparison between the MICs of some extracts and EOs of the same origin as O. syriacum
and C. winterianus from previous literature.

MICs of Extracts and EOs

Extracts Bacterial Isolates Fungal Isolates
Plant Reference
and EOs
E. coli K. pneumonia S. aureus A. niger C. albicans
Origanum EO 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL - -
[24]
Monolaurin EO >8 mg/mL >8 mg/mL 0.063 mg/mL - -
O. syriacum AE 12.5 µg/mL 6.25 C - - - [25]
O. syriacum EO - 40 µg/mL - - - [26]
C. khasianus EO 20 V 20 µg/mL 30 µg/mL - 100 µg/mL [27]
C. winterianus EO 0.98 µg/mL - 0.98 µg/mL - - [28]
C. winterianus EO 6 µg/mL - 2 µg/mL 2.5 µg/mL 1 µg/mL [29]
O. syriacum Combined AEs 2.5 mg/mL 2.5 mg/mL 2.5 mg/mL 5 mg/mL -
Current
and Combined EEs 2.5 mg/mL 1.25 mg/mL 5 mg/mL 5 mg/mL 2.5 mg/mL study
C. winterianus
Combined EOs 20% 5% 5% 5% 5%
MIC: minimum inhibitory concentration, EOs: essential oils, AE: Aqueous extract, EE: ethanolic extract, “-”:
not determined.
Foods 2024, 13, 1684 8 of 15

3.2. Time-Kill Results of the Aqueous and Ethanolic O. syriacum and C. winterianus Extracts and
EOs against the Bacterial and Fungal Isolates
The time-kill assay was performed to detect the time needed by the aqueous and
ethanolic O. syriacum and C. winterianus extracts and their EOs to exert their bacteriostatic
and bactericidal/fungicidal actions. In general, both aqueous and ethanolic extracts needed
at least 1 h to inhibit bacterial growth. Gram-positive bacteria were inhibited faster than
Gram-negative bacteria. The aqueous extracts inhibited bacterial growth within 2–4 h,
while the ethanolic extracts took 24 h to exert their inhibitory action. Similar to the aqueous
extracts and in contrast to the ethanolic extracts, the EOs inhibited bacterial growth within
only 1 h, with a better effect for the combined samples. For the fungal isolates, the mean
time needed for inhibition ranged between 1 and 4 h. The ethanolic extracts were faster in
inhibiting the growth of fungi than the aqueous extracts. However, the combined extracts
took more time to inhibit C. albicans and A. niger. The results of the time-kill assay against
the bacterial and fungal isolates are presented in Table 3 and Figures S3 and S4. In addition,
the significant p-values are presented in Table S2.

Table 3. Time-kill results of the aqueous and ethanolic O. syriacum and C. winterianus extracts and
their EOs against the bacterial and fungal isolates.

Time of Inhibition (h) of Bacterial and Fungal Isolates

Bacterial Isolates
Plant Extracts and Fungal Isolates
Oils Gram-Negative Bacteria Gram-Positive Bacteria
E. coli K. pneumonia C. freundii S. aureus S. intermedius C. albicans A. niger
C. winterianus AE 24 2 24 2 2 4 2
O. syriacum AE 24 1 2 2 2 1 1
Combination of AEs 1 1 1 2 24 2 1
C. winterianus EE 24 24 24 24 24 1 1
O. syriacum EE 24 24 24 24 24 2 1
Combination of EEs 24 24 24 24 24 2 2
C. winterianus EO 2 2 4 1 1 1 1
O. syriacum EO 1 2 4 24 2 2 2
Combination of EOs 1 4 1 1 2 4 4
EOs: essential oils, AE: Aqueous extract, EE: ethanolic extract.

3.3. Antibiofilm Activity of the Aqueous and Ethanolic O. syriacum and C. winterianus Extracts
and EOs against the Bacterial and Fungal Isolates
The antibiofilm assays were performed to determine the ability of the aqueous and
ethanolic O. syriacum and C. winterianus extracts and their EOs to inhibit and destroy
biofilms. In contrast to bacterial isolates, biofilms showed great resistance to both O. syri-
acum and C. winterianus extracts, as well as their EOs. Among the aqueous extracts, only
O. syriacum was able to inhibit the formation of S. aureus biofilm at a concentration of
0.3125 mg/mL. Similarly, among the ethanolic extracts, O. syriacum was able to inhibit the
formation of S. intermedius biofilm at a concentration of 0.3125 mg/mL and K. pneumonia
biofilm at a concentration of 0.3125 mg/mL. Unfortunately, among the combined extracts,
the ethanolic extract was only able to inhibit the formation of S. aureus biofilm at a con-
centration of 5 mg/mL. As for the fungal biofilms, the C. winterianus aqueous extract was
only able to inhibit the formation of C. albicans biofilm at a concentration of 0.625 mg/mL.
However, the A. niger biofilm was resistant to all extracts and EOs. The other extracts
and EOs did not show any inhibitory activity. The inhibitory percentages are presented in
Figure 2, and the significant p-values of the antibiofilm formation are presented in Table S3.
Foods 2024, 13, 1684 9 of 15

Foods 2024, 13, 1684 9 of 15

did not show any inhibitory activity. The inhibitory percentages are presented in Figure
2, and the significant p-values of the antibiofilm formation are presented in Table S3.

Figure 2. Inhibition of the formation of bacterial and fungal biofilms by the aqueous and ethanolic
Figure 2. Inhibition of the formation of bacterial and fungal biofilms by the aqueous and ethanolic
O. syriacum and C. winterianus extracts and their EOs (EO: essential oil).
O. syriacum and C. winterianus extracts and their EOs (EO: essential oil).
3.4.
3.4. Destruction
Destruction of of Pre-Formed
Pre-Formed Biofilms
Biofilms
Similar
Similar to the inhibition of
to the inhibition of biofilm
biofilm formation,
formation, thethe aqueous
aqueous and
and ethanolic
ethanolic O. O. syriacum
syriacum
and
and C.C. winterianus
winterianus extracts
extractsshowed
showedweak weakdestructive
destructiveactivity
activityagainst
against almost
almost allall
thethe bacte-
bacterial
rial biofilms.
biofilms. However,
However, the O.the O. syriacum
syriacum aqueousaqueous
extractextract
was ablewastoable to destroy
destroy the S.biofilm
the S. aureus aureus
biofilm at a concentration
at a concentration of 0.3125of 0.3125
mg/mL mg/mL andK.
and the thepneumonia
K. pneumonia biofilm
biofilm at aatconcentration
a concentration of
of 0.3125 mg/mL. As for the pre-formed fungal biofilms,
0.3125 mg/mL. As for the pre-formed fungal biofilms, the aqueous C. the aqueous C. winterianus extract
was able
able to
todestroy
destroythe theC.C.albicans
albicansbiofilm
biofilm only
only at aatconcentration
a concentration of 0.625
of 0.625 mg/mL.
mg/mL. How-
However,
ever,
A. nigerA. biofilm
niger biofilm
showed showed sensitivity
sensitivity to theto the combined
combined aqueousaqueous
extractextract at a concentra-
at a concentration of
tion
0.625ofmg/mL,
0.625 mg/mL,
as wellas aswell
to allasethanolic
to all ethanolic
extractsextracts at concentrations
at concentrations rangingranging
betweenbetween
2.5 and
2.5 and 5 mg/mL.
5 mg/mL. The destruction
The destruction percentages
percentages are presented
are presented Figure 3,Figure
and the3, significant
and the significant
p-values
p-values of the antibiofilm
of the antibiofilm formationformation are presented
are presented in Table S4.in Table S4.
Foods 2024,
Foods 13,13,
2024, 1684
1684 10 10
of of
15
Foods 2024, 13, 1684 10 of 15
15

Figure
Figure3. 3.
Destruction of of
Destruction thethe
pre-formed
pre-formed bacterial and
bacterial andfungal biofilms
fungal byby
biofilms thethe
aqueous and
aqueous ethanolic
and ethanolic
Figure
O.O.
syriacum3. Destruction
and C.C. of the pre-formed
winterianus extracts and bacterial
their EOsand
(EO:fungal biofilms
essential oil). by the aqueous and ethanolic
syriacum and winterianus extracts and their EOs (EO: essential oil).
O. syriacum and C. winterianus extracts and their EOs (EO: essential oil).
3.5. Preservation of of
3.5. the Table Grapes byby thethe
Prepared Film
3.5. Preservation
Preservation of the
the Table
Table Grapes
Grapes by the Prepared
Prepared FilmFilm
The appearance
The appearance of
The appearance of grape
ofgrape berries
grapeberries was
berrieswas improved
wasimproved
improved inin
in terms
terms
terms ofof
of brightness
brightness
brightness and
and andcolor
atat
color
color at
the
the
thebeginning
beginning
beginning of the shelf
of the
of the life
shelf
shelf (1st
lifelife (1st
(1st day).
day).
day). The best
Thebest
The external
bestexternal appearance
externalappearance
appearanceof of the
ofthe grape
the grapebunches
grape bunches
bunches
after
after
afterhaving
having
havingbeen stored
been
been stored
storedfor 3737
for
for days
37 days
daysininthe
in refrigerator
the
the refrigerator
refrigerator atat4 °C
at was
44 °C
◦C wasinin
was the
in bunches
the
the bunches
buncheswrapped
wrapped
wrapped
inin
thethepackaging
packaging prepared
prepared with
with the
theaqueous
aqueous extract
extract of C.
of winterianus
C. winterianus
in the packaging prepared with the aqueous extract of C. winterianus at a concentration at a
at concentration
a concentration ofof
of
1010
mg/mL.
10 mg/mL. This gives
This givesthe
thegrape
grape clusters
clusters the ability
the ability to maintain
to maintain skin
skin
mg/mL. This gives the grape clusters the ability to maintain skin consistency comparedconsistency
consistency compared
compared
toto
thetherest ofof
rest the clusters,
the clusters,where
where thetheberries
berries lose
lose their firmness
their firmness and
andbecome
become wrinkled.
wrinkled.The
The
preservation results are presented
presented in
preservation results are presented in Figure
in Figure 4.
Figure 4. 4.

Figure
Figure4. 4.
Appearance
Appearanceofof
Appearance the
of grapes’
the
the structures
grapes’
grapes’ after
structures
structures packaging
after
after with
packaging
packaging thethe
with
with the aqueous
C.C.
aqueous
aqueous winterianus
C. ex-ex-
winterianus
winterianus extracts.
tracts.
tracts.
Foods 2024, 13, 1684 11 of 15

4. Discussion
In the framework of studying the antibacterial and antifungal activity of C. winterianus
and O. syriacum extracts and their EOs, they were tested against five bacterial isolates
(E. coli, K. pneumonia, C. freundii, S. aureus, and S. intermedius) isolated from urine samples
and two fungal isolates (C. albicans and A. niger) isolated from spoiled food samples. The
mentioned extracts and their EOs proved to be effective antimicrobial agents against
bacteria and fungi [19]. However, the antimicrobial activity of the combined C. winterianus
and O. syriacum extracts and EOs has not been taken into consideration previously. So,
this study aimed to check the antimicrobial efficacy of the combined C. winterianus and
O. syriacum extracts, as well as their EOs. C. winterianus and O. syriacum extracts and their
EOs, like other natural extracts and oils, were proved to be not toxic, safe, and friendly to the
environment [19,29,30]. This study revealed that C. winterianus and O. syriacum extracts and
their EOs, especially their combinations, exhibited significant antibacterial and antifungal
activities. The aqueous and ethanolic C. winterianus and O. syriacum extracts exhibited
bacteriostatic activity, while the EOs had mostly bactericidal action against the tested
isolates. This antimicrobial efficacy is caused by the presence of phenolic and monoterpene
components in the extracts and the EOs, especially carvacrol and thymol [13,30]. Carvacrol
was proved to be the most effective component against microbes. Thymol and carvacrol
were proved to exert toxic effects, especially against E. coli and S. aureus, with a stronger
action by carvacrol [14].
In addition, these bactericidal and bacteriostatic activities could be due to the hy-
drophobic nature of the extracts and EOs, which facilitate their penetration into the bacterial
cell, thus changing the orientation of lipids in the cell membrane [14]. These changes alter
the chemical and physical properties of the cell membrane, which increases the electron
flow and proton flux across the membrane, thus coagulating the cell contents and causing
cell death [14,19]. Among the tested bacterial isolates, S. aureus was the most sensitive
bacterium and E. coli was the most resistant. This means that C. winterianus and O. syriacum
extracts and EOs exerted better inhibitory activity against Gram-positive bacteria than
Gram-negative bacteria. Gram-negative bacteria are more resistant than Gram-positive
bacteria due to the presence of an outer membrane rich in lipopolysaccharides in the
Gram-negative bacterial cell wall [19,31,32].
For fungal susceptibility, O. syriacum extract and its EO were proved to show efficacy
against many fungal isolates [16]. The mechanism of action is similar to that of bacteria.
For example, Daouk et al. reported that the presence of thymol and carvacrol in O. syriacum
extracts plays a vital role in the antimicrobial activity of the extract as well as the EO,
especially against Penicillium and Fusarium [16]. The cytotoxic activity was attributed
to their lipophilic nature, which enables them to penetrate the cell membrane of fungi
leading to damage of the cellular components. In addition, the penetration of the EOs
into the cell leads to interaction with cellular ions, leading to depletion of the ATP pool
and the leakage of calcium, magnesium, and potassium ions, thus damaging the cells
and causing cell lysis [16,19]. Another study reported the effective antifungal activity of
O. syriacum and many species of Citronella EOs against A. niger. They inhibited the mycelial
growth of fungi [16,17]. This effect was specifically shown to be due to the presence of
carvacrol and thymol, in addition to many other compounds including sesquiterprenoids,
monoterpenoids, eudesmol, geraniol, and geranyl acetate [17]. The mentioned compounds
were proved to work in a cascade of reactions. Dangol et al. reported some antagonistic
mechanisms including the disruption of the cytoplasmic membrane leading to leakage
and the inhibition of sporulation, thus leading to cell lysis [17]. Other mechanisms include
changes in the permeability and damaging the cell membrane, inhibition of the respiratory
metabolism, forming chimeras with the DNA (especially carvacrol), degenerating the cell
morphology, and decreasing the total protein amount [33,34]. The proposed mechanisms
of the antimicrobial actions are illustrated in Figure 5.
It is worth mentioning that the action of the extracts, as well as their EOs, depends
on their chemical composition. As mentioned previously, O. syriacum and C. winterianus
 Foods 2024, 13, 1684 12 of 15

are rich in phenolic compounds. Previous literature reported that carvacrol, thymol,
cymene, terpinene, myrcene, caryophyllene, and thymoquinones are the main constituents
of O. syriacum and C. winterianus extracts [26,33,34]. For example, Mesmar et al. [15]
listed the mentioned chemical compounds and reported that they are responsible for the
antibacterial and antifungal mechanisms exerted by the extracts.
In addition, the chemical composition of the EOs plays a vital role in their antibacterial
and antifungal activities. Rammal et al. showed in a recent study that the main chemical
compounds revealed in the O. syriacum and C. winterianus EOs include geraniol and
citronellol, along with many other compounds found in lower percentages. The compounds
are responsible for the antibacterial and antifungal mechanisms exerted by the O. syriacum
and C. winterianus EOs.
The time-kill results revealed that all isolates required 1 to 4 h to be inhibited. Usually,
bacteria need at least 12 h for maturation and fungi need about 48 h for full growth.
However, both bacteria and fungi are able to fix to their surface within 4 h. This time is
needed for nutrient uptake. This means that the extracts and their oils inhibited the nutrient
uptake by bacteria and fungi, thus inhibiting their attachment and growth [21,22].
Regarding the antibiofilm results, unfortunately, the C. winterianus and O. syriacum
extracts and their EOs had a very weak inhibitory activity against almost all biofilms. They
neither inhibited the biofilm formation nor destroyed the pre-formed biofilms. However,
the exerted inhibitory activity against some biofilms is explained by the fact that natural
extracts and EOs are able to interact with the exopolysaccharides secreted by biofilms to
prevent their attachment [19,35,36]. In addition, the inhibitory concentrations recorded
were independent of the MICs and MBCs/MFCs. This is attributed to the fact that anti-
biofilm activities and concentrations are independent due to the variation in absorbances
and cell enumeration [37]. The interaction between the major components of the extracts
and EOs leads to inhibitory activities. However, the weak activity could be explained by
024, 13, 1684 13 of 15
the fact that biofilms create more resistance due to the formation of clusters of cells making
the bacteria and fungi stronger [35–37].

Figure 5. Proposed mechanisms

Figure of action
5. Proposed of extracts
mechanisms against
of action of bacteria (createdbacteria
extracts against with BioRender.com,
(created with BioRender.com,
accessed on 16 May 2024) modified from Liang et al. [38].
accessed on 16 May 2024) modified from Liang et al. [38].

5. Conclusions Grapes are non-climacteric perishable fresh food products with several other post-
harvest storage problems such as loss of firmness, berry dropping, stem discoloration, and
This studydesiccation.
reported theC.antibacterial and antifungal
winterianus extract was ablepotentials
to protectofthe
C. winterianus
skin of the and
berries more than
O. syriacum extracts and their EOs against various uropathogenic bacteria and foodborne
fungi. In addition, their preservative strength was tested on grapes. For this, their antibac-
terial activity was detected against five bacteria isolated from urine (E. coli, K. pneumonia,
C. freundii, S. aureus, and S. intermedius). Both the aqueous and ethanolic O. syriacum and
C. winterianus extracts exhibited bacteriostatic and bactericidal actions against the tested
Foods 2024, 13, 1684 13 of 15

O. syriacum extract. We can consider this film as a concentration of polyphenols including

flavones, anthocyanins (both coloring pigments), and stilbenes such as resveratrol. In
nature, polyphenols have essential missions for survival. In addition to their defense
against environmental attacks (ultraviolet radiation, invasion by insects, fungi, and viruses),
polyphenols also perform “appetizing” functions. They are partly responsible for the
sensory and nutritional qualities of plants including astringency, color, and odor. In the
flavonoid family, anthocyanins are responsible for the red color of grapes; resveratrol, in
its natural combination in whole grapes, has antifungal properties. It may be that the
C. winterianus extract was able to preserve them.
Overall, the C. winterianus and O. syriacum extracts and their EOs had significant
antibacterial and antifungal activities. These effects allow for their use in food preservation
and protection against bacterial and fungal spoilage.

5. Conclusions
This study reported the antibacterial and antifungal potentials of C. winterianus and
O. syriacum extracts and their EOs against various uropathogenic bacteria and foodborne
fungi. In addition, their preservative strength was tested on grapes. For this, their antibac-
terial activity was detected against five bacteria isolated from urine (E. coli, K. pneumonia,
C. freundii, S. aureus, and S. intermedius). Both the aqueous and ethanolic O. syriacum and
C. winterianus extracts exhibited bacteriostatic and bactericidal actions against the tested
isolates. Furthermore, the EOs showed better inhibitory activity than the extracts on the
same isolates. Their action was exerted within 2 to 4 h of incubation with the bacteria.
However, the extracts and EOs exerted weak antibiofilm activity against the bacterial
biofilms. They were neither able to inhibit the formation of the biofilms nor eradicate the
pre-formed biofilms. For the antifungal activities, two foodborne fungi isolated from lemon
and jam were tested (A. niger and C. albicans). The O. syriacum and C. winterianus extracts
and EOs exerted fungistatic actions against the tested fungi. However, the EOs exerted
a stronger action than the extracts. Their action was reported after 3 to 4 h of incubation.
Their antibiofilm action against the fungal isolates was relatively weak. For the packaging
potential, the O. syriacum and C. winterianus extracts were able to preserve the grapes
for about 40 days, with a better effect for C. winterianus. It is worth mentioning that the
combined samples of the two extracts and their EOs had better potential against bacteria
and fungi than the individual O. syriacum and C. winterianus. This study opens an avenue
for the use of combined O. syriacum and C. winterianus extracts and EOs against different
uropathogenic and foodborne microorganisms and their use in food preservation.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/foods13111684/s1.
Author Contributions: Conceptualization, M.I.K.; data curation, M.R. and M.M.; formal analysis,
M.R., S.K., M.M., C.H., M.I.K. and E.B.; funding acquisition, M.B. and M.I.K.; investigation, S.K., M.M.
and C.H.; Methodology, M.M., M.I.K. and M.H.E.-D.; project administration, M.H.E.-D.; resources,
M.B. and M.H.E.-D.; supervision, M.I.K., E.B. and M.H.E.-D.; validation, M.R., S.K., A.B., M.M.,
M.B., C.H., M.I.K., E.B. and M.H.E.-D.; visualization, A.B. and M.B.; writing—original draft, M.R.,
S.K., M.M. and M.I.K.; writing—review and editing, A.B., M.B., C.H., E.B., M.I.K. and M.H.E.-D. All
authors have read and agreed to the published version of the manuscript.
Funding: This project was supported by funding from Petra University.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The original contributions presented in the study are included in the
article/Supplementary Materials, further inquiries can be directed to the corresponding authors.
Conflicts of Interest: The authors declare no conflicts of interest.
Foods 2024, 13, 1684 14 of 15

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