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1/15Brazilian Journal of Biology, 2024, vol. 84, e286731 | https://doi.org/10.1590/1519-6984.286731
Original Article
THE INTERNATIONAL JOURNAL ON NEOTROPICAL BIOLOGY
THE INTERNATIONAL JOURNAL ON GLOBAL BIODIVERSITY AND ENVIRONMENT
ISSN 1519-6984 (Print)
ISSN 1678-4375 (Online)
This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Ethanolic extracts of seasonally collected natural bee products (honey, propolis, royal jelly (RJ), and bee venom (BV))
were tested for their potential as antimicrobial agents against antibiotic-resistant bacteria and fungi. These extracts
exhibited various inhibitory effects on antibiotic-resistant bacteria (Streptococcus pneumoniae, Staphylococcus
aureus, MRSA, Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus
vulgaris, and Haemophilus influenzae) and fungi (Aspergillus brasiliensis and Candida albicans), with the exception
of S. pneumonia, which was not inhibited by honey and RJ extracts, and P. aeruginosa, which was not inhibited
by RJ extracts. Interestingly, extracts of BV and its major content, melittin (MEL), displayed a wide spectrum of
antimicrobial activity against all tested bacteria and fungi. This is the first study to show that propolis extract has
bactericidal activity against S. pneumoniae and that BV extract and MEL have antibacterial activity against P. vulgaris,
H. influenzae, and H. influenzae type b. Extracts of bee products collected in the spring generally exhibited the most
significant antibacterial and antifungal activities. Based on total phenolic content (TPC) and total flavonoid content
(TFC), it was found that spring samples of propolis, RJ, and honey, in that order, were the richest. Also, LC-MS-MS
analysis of MEL content in BV demonstrated that it was the highest in spring sample. In terms of MIC and MBC
values, Gram-positive bacteria were the most susceptible to bee products. First and foremost, the antimicrobial
activity of bee products was ranked in descending order based on MIC values: BV, MEL, propolis, RJ, and honey.
Keywords: antimicrobial, MIC, royal jelly, bee venom, Melittin.
Resumo
Extratos etanólicos de produtos apícolas naturais coletados sazonalmente (mel, própolis, geleia real (GR) e veneno
de abelha (VA)) foram testados quanto ao seu potencial como agentes antimicrobianos contra bactérias e fungos
resistentes a antibióticos. Esses extratos demonstraram vários efeitos inibitórios sobre bactérias resistentes a
antibióticos (Streptococcus pneumoniae, Staphylococcus aureus, MRSA, Salmonella typhimurium, Escherichia coli,
Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus vulgaris e Haemophilus influenzae) e fungos (Aspergillus
brasiliensis e Candida albicans), com exceção de S. pneumonia, que não foi inibida por extratos de mel e GR, e
P. aeruginosa, que não foi inibida por extratos de GR. Curiosamente, os extratos de VA e seu principal conteúdo,
melitina (MEL), apresentaram um amplo espectro de atividade antimicrobiana contra todas as bactérias e fungos
testados. Este é o primeiro estudo a mostrar que o extrato de própolis tem atividade bactericida contra S. pneumoniae,
e que o extrato de VA e MEL têm atividade antibacteriana contra P. vulgaris, H. influenzae e H. influenzae tipo
b. Extratos de produtos apícolas coletados na primavera geralmente exibiram as atividades antibacterianas e
antifúngicas mais significativas. Com base no conteúdo fenólico total (TPC) e no conteúdo total de flavonoides
(TFC), verificou-se que as amostras de primavera de própolis, GR e mel, nessa ordem, foram as mais ricas. Além
disso, a análise LC-MS-MS do conteúdo de MEL em VA demonstrou que foi o mais alto na amostra de primavera.
Em termos de valores de MIC e MBC, as bactérias gram-positivas foram as mais suscetíveis aos produtos apícolas.
Em primeiro lugar, a atividade antimicrobiana dos produtos apícolas foi classificada em ordem decrescente com
base nos valores de MIC: VA, MEL, própolis, GR e mel.
Palavras-chave: antimicrobiano, MIC, geleia real, veneno de abelha, Melitina.
Antimicrobial activities of seasonally collected bee products:
honey, propolis, royal jelly, venom, and mellitin
Atividades antimicrobianas de produtos apícolas coletados sazonalmente: mel,
própolis, geleia real, veneno e melitina
M. Obeidata* , M. A. Haddadb and S. A. Ghnamatc
aAl-Balqa Applied University, Faculty of Science, Department of Medical Laboratory Sciences, Al-Salt, Jordan
bAl-Balqa Applied University, Faculty of Agricultural Sciences, Department of Nutrition and Food Processing, Al-Salt, Jordan
cAL-Balqa Applied University, Faculty of Agricultural Technology, Department of Plant Production and Protection, Al-Salt, Jordan
*e-mail: obeidat@bau.edu.jo
Received: May 19, 2024 – Accepted: September 18, 2024
Brazilian Journal of Biology, 2024, vol. 84, e2867312/15
Obeidat, M., Haddad, M.A. and Ghnamat, S.A.
inflammatory activities of propolis are due to its content
of phenolic compounds and their esters, flavanone, and
flavone (Bankova, 2005).
Bee venom is a complex combination of compounds
that make up a mixture of proteins, peptides, amino
acids, phospholipids, carbohydrates, biogenic amines,
pheromones, volatile compounds, and a high portion of
water (Pascoal et al., 2019). Dried BV is composed of ~40%
Melittin (MEL), which is considered the most important
water-soluble cationic peptide of BV and has a sequence
of 26 amino acid residues (Ceremuga et al., 2020). It is
produced by bees when they feel threatened and is often
created in defense against predators. A lot of evidence
indicates that humans have historically used BV in the
treatment of many diseases and as an antimicrobial peptide
(Memariani et al., 2019). Among the popular applications
of bee stings is for joint and bone pain by placing the bees
on the areas where the patient suffers (Zhang et al., 2018).
It was reported that BV exhibited antimicrobial effects
against bacteria, fungi, and viruses (Abd El-Wahed et al.,
2019), in addition to its cytolytic activity on the membranes
of cells, including cancerous cells (Pascoal et al., 2019).
In this study, the effectiveness of the antimicrobial
activity of various natural bee products was compared
seasonally. Moreover, there has been no previous survey
on BV from Jordan, specifically identifying the MEL content
of the toxin and its potential antibacterial and antifungal
activities. To the best of our knowledge, so far there are
no studies seasonally comparing the antimicrobial effects
of honey, RJ, propolis, BV, and MEL on Gram-positive and
Gram-negative bacteria as well as fungi. Therefore, the
current study was initiated to investigate the antimicrobial
activities of seasonally collected bee products from
Jordan, including; honey, RJ, propolis, and BV, as well as
its main constituent (MEL), toward pathogenic bacteria
and fungi and to determine their minimum inhibitory
concentration (MIC).
2. Materials and Methods
2.1. Bee products collection
All bee products of Apis mellifera used in this study
were collected from areas located in Al-Anabtawi apiaries,
which are located in the middle of Jordan (32°08’52.4”N,
35°50’52.0”E) about 15 km from the capital Amman.
The apiaries are allocated in a mountainous region that
is rich in flora and characterized by cold weather in
the winter, moderate weather in spring and autumn,
and moderate to high temperature in the middle of the
summer season. The collection process was carried out
in mid-April, mid-July, and mid-October, spanning three
seasons; spring (March-May), summer (June-August),
and autumn (September-November). Nine honey samples
were collected from three honey bee colonies in mid-April
(three samples), mid-July (three samples), and mid-October
(three samples). Also, samples of RJ, propolis, and BV were
collected in the same manner.
For the BV collection, an in-house device was designed
to mimic the principle of a bee stinging without harming
1. Introduction
The ineffectiveness of antibiotics in treating severe
infectious bacterial and fungal diseases has become a
global problem that creates a pharmaceutical challenge.
Recent studies (CDC, 2019; Frieri et al., 2017) have
shown that antibiotics are less effective or insufficient
in eliminating certain forms of frequent pathogens of
bacteria and fungi that exhibited multidrug resistance to
almost all antibiotics. Therefore, the continued threat of
antibiotic resistance demanded that scientists search for
new antimicrobial molecules from new natural sources to
avoid microbial infections from becoming dangerous. Many
natural bioactive compounds, produced by Apis mellifera
honeybees, have the potential to induce antimicrobial
effects. Natural bee products such as honey, royal jelly
(RJ), propolis, wax, and bee venom (BV) are the ancient
source of treatment for many pathologies and symptoms
and have been used in several products and drugs.
Honey has historically been used by the ancient
Egyptians to heal wounds and prevent infections (Majno,
1975). In addition, honey is described as a source of
healing in the holy Quran (16:68–69), which mentions that
honeybees produce from their bellies a drink of varying
colors that is healing for men. It is rich in sugars (~80%;
fructose, glucose, sucrose, and some disaccharides), has
~20% water content, and small quantities of proteins,
amino acids, vitamins, enzymes such as glucose oxidase
(GOx), polyphenols, and minerals (Cooke et al., 2015;
Pasupuleti et al., 2017). Honey has antibacterial, antifungal,
and antiviral properties; the antimicrobial activity of honey
is multifactorial and it is mainly due to the production
of hydrogen peroxide (H2O2) via glucose oxidase (GOx),
honey’s acidity (pH ~3.9), defensin-1 (Def-1) produced
from bee’s hypopharyngeal gland, the non-enzymatic
conversion of dihydroxyacetone to methylglyoxal (MGO),
low water activity (high osmotic pressure), and various
phenolic compounds (Yupanqui Mieles et al., 2022).
Royal jelly is a slightly acidic, milky, viscous secretion
produced by nurse bees with a bitter taste and pungent
odor (Fujita et al., 2013). It is fed to honeybee larvae,
especially during the first three days, and to the queen bee
throughout its lifetime. In addition, RJ has been extensively
acknowledged to exhibit different pharmacological
activities, including; antimicrobial, antitumor, antioxidant,
immune-inducing, anti-inflammatory, vasodilative,
hypotensive, and anti-hypercholesterolemic agents
(Eshraghi and Seifollahi, 2003; Nagai and Inoue, 2004;
Ramadan and Al-Ghamdi, 2012; Ramanathan et al., 2018;
Seven et al., 2014). The antimicrobial effects of RJ are due
to its fatty acids content (Melliou and Chinou, 2005) and its
constituent of proteins and peptides (Bílikova et al., 2015).
Propolis is collected by bees, and it is a resinous
compound composed of resins, vegetable balsam, wax,
essential and aromatic oils, pollen, and organic debris
(Burdock, 1998). It is a potent natural bee product that
is used for different medical benefits, including its
effects as an antibacterial, antifungal, antiviral, and anti-
inflammatory agent (Bankova, 2005; Martins et al., 2002).
One of the propolis functions within hives is to protect
against bacteria and fungi. The antimicrobial and anti-
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Antimicrobial activities of bee products
or killing them. The BV collector wires are about three
millimetres (mm) in diameter, have a battery voltage of 12-
15 volts, and generate electrical impulses with frequencies
ranging from 50 to 1000 Hz. The wires were cleaned daily.
The BV samples were collected from three colonies using
an in-house developed system that is installed in the
beehives and ensures that a certain number of bees are
trapped in their frames. The process takes half an hour per
colony. After the venom collection process, the yellowish,
gum-like venom instantly dries up and turns into a white-
yellowish crystal form on the glass plates. The formed
crystals were then cut into containers and preserved at
-20°C in a dark, dry environment to avoid autolysis by the
protease present in BV. A quantity of approximately 10 g
of dry BV was collected.
2.2. LC-MS-MS analysis of bee venom
Liquid chromatography with tandem mass spectroscopy
(LC-MS-MS) (Sceix 3200, USA) was used. Analytical
standards of MEL (the most important and major
compound of BV), BV, acetonitrile, trifluoroacetic acid
(TFA), and 0.22 µm filter membrane were used. Stock
standard solutions were prepared to achieve 100 ppm.
The calibration curve was performed at 4 points of standard:
10 ppm, 25 ppm, 50 ppm, and 100 ppm. The seasonally
collected three BV samples were weighted and then they
were diluted to have a final concentration of 1 mg/ml.
LC-MS-MS studies were carried out using AB Sciex 3200;
the following elements were used: MS detector, 25°C oven
temperature, chromatographic column: ACE C8 (50mm x
2.1) 5 µm, separation temperature: 25°C, 1, 2, and 2.0 ml/
min flow rates for the mobile phase. Following eluents as
a basis for A solvent, 0.5% mM ammonium chloride with
0.1% formic acid A, and 0.1% acetonitrile in solution for B
air, water, and acetonitrile. Daughter fragment 712 was
used in the LC-MS-MS.
2.3. Preparation of bee products’ extracts and melittin
Seasonally collected samples of propolis were processed
into a soft powder. The collected RJ and BV samples were
evaporated and processed by a lyophilizer (Heto Dry
Winner / Thermofisher, USA) to make a powder to enhance
stability by reducing the breakdown rate of lipid and
peptide contents. Honey samples were dried by the vacuum
drying process. Thereafter, propolis powder, vacuum-dried
honey, as well as freeze-dried RJ and BV samples (three
each), were extracted via 70% ethanol (1:10, w/v) for one
week at room temperature with shaking at 150 rpm.
The extracts were filtered through Whatman No. 1 filter
paper. Afterward, the filtrates were evaporated until dry.
The standard MEL (Gene script, South Korea) was a white
lyophilized powder. The dried extracts and the standard
MEL were dissolved in 0.05% dimethyl sulfoxide (DMSO) at
400 mg/ml final stock concentration, respectively. All stock
solutions were purified by filtration through 0.22 µm filter
units and kept at -20°C until use.
2.4. Assessment of total phenolic content
The total phenolic content (TPC) in honey, RJ, and
propolis samples collected in spring was estimated as
previously described (El-Guendouz et al., 2016) with a
slight alteration. A volume of 50 µl of each sample stock
solution was thoroughly mixed with 250 µl of 0.2 N Folin-
Ciocalteu’s reagent for 5 minutes. Then, 200 µl aliquot of
7.5% Na
2
CO
3
were added. After incubation of all samples at
room temperature for 2 hr, the absorbance was measured at
760 nm. Distilled water in place of bee products was used
as a blank. The TPC was calculated from the calibration
curve as mg of Gallic acid equivalents per gram of sample
(mg GAE/g sample) using a 0.04-1.00 mg/ml concentration
range of Gallic acid.
2.5. Assessment of total flavonoid content
The total flavonoid content (TFC) in honey, RJ, and
propolis samples collected in springtime was determined
according to the method performed by El-Guendouz et al.
(2016). Each sample stock solution received an equal volume
of 200 µl of 20% AlCl3. The mixtures were left for 1 hr at
room temperature, then the absorbance was measured
at 420 nm. The TFC was expressed as mg of Quercetin
equivalents per gram of bee product (mg QE/g sample)
using a calibration curve with a concentration range of
0.04-1.00 mg/ml of equivalents.
2.6. Test strains of bacteria and fungi
Five Gram-positive bacteria (Streptococcus pneumoniae
ATCC 6305, Staphylococcus aureus ATCC 25923, Methicillin-
resistant Staphylococcus aureus ATCC 95047 (MRSA), and
two clinical strains; S. pneumoniae and S. aureus), seven
Gram-negative bacteria (Salmonella typhimurium ATCC
14028, Escherichia coli ATCC 8739, Pseudomonas aeruginosa
ATCC 27253, Klebsiella pneumoniae ATCC 7700, Proteus
vulgaris ATCC 33420, and two clinical strains; Haemophilus
influenzae and H. influenzae type b), and four fungi
(Aspergillus brasiliensis ATCC 16404, Candida albicans ATCC
10231, and two clinical strains; A. brasiliensis, C. albicans)
were used to determine the antimicrobial activities of
bee products. All bacterial and fungal strains used in
the current study exhibited various forms of antibiotic
resistance (Obeidat et al., 2017).
2.7. Assessment of antimicrobial activity
Crude extracts of honey, RJ, and propolis (diluted to
200 mg/ml) as well as BV extracts and MEL (diluted to 10 mg/
ml) were screened for antimicrobial activities using the
agar-well diffusion method that was previously described
(Perez, 1990) with some alterations (Obeidat et al., 2017).
In brief, 50 µl aliquots from each bacterial species and
fungal species were evenly swabbed on Mueller-Hinton
agar medium (MHB) and Sabouraud dextrose agar
medium (SDA), respectively, and allowed for 5 min to
dry. A sterile cork borer was used to make wells, 6 mm in
diameter, in the seeded medium. Then, 50 µl from each
diluted stock solution of bee product extract was added
into each well, parallel to wells containing 50 µl DMSO
that serve as negative controls, and left on the bench for
1 hr for proper diffusion, and subsequently incubated at
37°C for 24 h and at 28°C for 48 h for bacteria and fungi,
respectively. To determine the antimicrobial activities, the
diameter of inhibition zones generated was measured and
Brazilian Journal of Biology, 2024, vol. 84, e2867314/15
Obeidat, M., Haddad, M.A. and Ghnamat, S.A.
expressed as mean±SD of triplicates. The disk diffusion
method was used for screening bacteria for multidrug
resistance to seven standard antibiotics; ampicillin (10µg),
chloramphenicol (30 µg), erythromycin (15 µg), nalidixic
acid (30 µg), penicillin G (10 units), streptomycin (10 µg),
and vancomycin (30 µg). For fungi, the resistance was also
determined to cycloheximide (250 µg) and nystatin (10 µg).
2.8. Minimum inhibitory concentration
The minimum inhibitory concentration (MIC), the
minimum bactericidal concentration (MBC), and the
minimum fungicidal concentration (MFC) were evaluated
for natural bee products that showed significant
antimicrobial activity (i.e., collected in springtime). The MIC,
MBS, and MFC were determined as previously described
by Nakamura et al. (1999) and Dulger and Aki (2009), with
some alterations reported by Obeidat (2011). The bacterial
cultures were grown in Mueller-Hinton broth (MHB) for
24 h at 37°C and adjusted to 1 x 108 CFU/ml. The fungal
cultures, on the other hand, were grown for 24 h at 30°C
and adjusted to 1 x 107 spore/ml in Sabouraud dextrose
broth (SDB). A dilution series of each bee product extract
was prepared. An aliquot of 100 µl from each extract
dilution was transferred to a 96-well microplate well that
had previously received 900 µl of the adjusted cultures of
the test microorganisms. The final concentrations were in
the range of 128 mg/ml to 1 mg/ml for ethanolic extracts
of honey, propolis, and RJ and in the range of 2048 µg/
ml to 1 µg/ml for BV extract and MEL. The microplates
were incubated for 24 h at 37 and 30°C for bacteria and
fungi, respectively. The values of MIC, MBC, and MFC were
determined by plating 50 µl from clear wells onto MHA for
bacteria and SDA for fungus. The MIC was considered to
be the lowest concentration in the sample that prevented
visible growth of bacteria or fungi. The MBC or MFC was
defined as the lowest concentration that yields negative
subcultures or only one colony of bacteria or fungi.
All samples were examined in triplicate.
2.9. Statistical analysis
The results of all generated inhibition zone diameters
were presented as the mean ± standard error (SE).
To compare mean zone of inhibition values, one-way
analysis of variance (ANOVA) followed by Tukey’s test
was applied using the IBM SPSS Statistics 19.0 program
for Windows. A P value of less than 0.05 was considered
statistically significant.
3. Results
The mass spectrometry experiment was performed
in full scan mode on the spring, summer, and autumn
samples of BV and revealed the presence of MEL in all BV
samples as a major peptide. Figure 1 illustrated that MEL
spikes were detected at various concentrations and have
a retention time (RT) of 4.5 during the spring, summer,
and autumn periods.
The TPC and TFC levels in ethanolic extracts of honey,
RJ, and propolis collected from Apis mellifera bees in
springtime were significantly the highest and respectively
comprised 0.64±0.02, 29.30±1.54, and 168.81±6.19 mg GAE/g
for TPC; and 0.048±0.002, 2.29±0.12 and 84.94±2.64 mg
QE/g for TFC (Table 1). On the other hand, the assessed
TFC values of bee products were lower than those of TPC.
It was observed that the extracts of bee products were
slightly acidic (propolis and BV) to acidic (honey and RJ).
The results of LC-MS-MS demonstrated that there are
different MEL concentration percentages due to variations
in seasons of collection, and the highest concentration of
MEL (29.75±4.17%) was observed in the collected BV during
spring, whereas the lowest concentration (14.50±4.03%)
was observed in the summer sample (Table 1).
The antibacterial activity of natural bee products was
determined against Gram-positive and Gram-negative
bacteria at concentrations of 200 mg/ml (honey, RJ, and
propolis) and 10 mg/ml (BV and MEL). Table 2 illustrated
that honey extracts showed antibacterial activity against
all antibiotic-resistant Gram-positive bacteria examined
in this study except the clinical strain S. pneumoniae.
Moreover, honey extract of the spring sample exhibited
significant antibacterial activity against S. pneumoniae ATCC
6305, MRSA ATCC 95047, and the clinical strain S. aureus.
Similarly, honey extract demonstrated antibacterial
Figure 1. Detection of melittin (MEL) spikes in the crude bee venom
(BV) by LC-MS-MS for BV samples collected in spring (A), summer
(B), and autumn (C), retention time (RT) = 4.5 min.
Brazilian Journal of Biology, 2024, vol. 84, e286731 5/15
Antimicrobial activities of bee products
Table 1. Total phenolic and flavonoid contents of honey, royal jelly, and propolis, as well as melittin content in venom samples collected
from Apis mellifera bees in spring.
Bee product Season Moisture% pH TPCa
(mg GAE/g)
TFCb
(mg QE/g) Melittin%
Honey Spring 16.53±2.29c3.75±0.02b0.64±0.02b0.048±0.002b-
Summer 16.49±1.87c3.71±0.11b0.57±0.03a0.041±0.004a-
Autumn 18.96±3.17c3.73±0.04b0.50±0.06a0.035±0.010a-
Royal Jelly Spring 67.50±3.78d3.32±0.06a29.30±1.54d2.29±0.12d-
Summer 66.35±2.01d3.29±0.03a25.34±0.67c2.21±0.05d-
Autumn 66.30±2.67d3.33±0.04a23.48±1.33c1.98±0.11c-
Propolis Spring - 5.13±0.07e168.81±6.19f84.94±2.64g-
Summer - 5.07±0.04de 150.11±11.17e65.09±5.77f-
Autumn - 5.09±0.02e148.39±9.23e54.36±3.83e-
Venom Spring 4.74±0.67a4.93±0.12cd - - 29.75±4.17c
Summer 6.89±1.33b4.87±0.09c- - 14.50±4.03a
Autumn 14.69±1.78c5.01±0.02d- - 21.54±2.98b
Results are Means±SE of triplicates. Values within a column followed by different letters are significantly different by Tukey’s studentized
range test (α = 0.05). aTPC, Total phenolic content; GAE, Gallic acid equivalents; bTFC, Total flavonoid content; QE, Quercetin equivalents.
Table 2. Antibacterial activity of honey, royal jelly, propolis, venom, and melittin that are seasonally collected from Apis mellifera bees
against pathogenic Gram-positive bacteria.
Inhibition Zone (mm)a
S
.
pneumoniae
ATCC 6305
S
.
aureus
ATCC 25923
MRSAb
ATCC 95047
S
.
pneumoniae S
.
aureus
Antibiotic
Resistancec
AMP10, P10,
VA30
AMP10, ERY15,
P10, VA30
AMP10, P10 AMP10, CHL30,
ERY15, NA30,
P10, S10, VA30
AMP10, ERY15,
P10, S10, VA30
Bee productdSeason
Honey Spring 23.67±2.08e14.00±2.00c18.67±1.15d0a28.33±2.73e
Summer 15.33±0.58c17.67±1.15d13.67±1.53c0a26.67±2.89e
Autumn 10.33±0.58b13.33±2.73bc 9.00±2.00b0a18.33±0.58d
Royal Jelly Spring 12.67±2.89bc 12.00±1.00bc 13.67±1.15bc 0a21.33±0.58d
Summer 11.33±1.53bc 11.67±2.89bc 10.67±2.08bc 0a21.00±2.00d
Autumn 8.67±3.06b10.33±2.73bc 0a0a20.33±2.08d
Propolis Spring 16.33±0.58e12.67±1.53c17.67±2.08e9.33±1.53b22.67±2.89f
Summer 15.33±2.73cde 11.33±2.73bcd 15.67±1.15de 7.67±2.08b21.00±1.00f
Autumn 12.33±2.73bcd 12.33±2.73bcd 15.33±0.58de 0a18.33±2.73e
Venom Spring 32.67±3.06g27.67±1.15f25.67±1.15e23.67±2.08cde 38.33±0.58h
Summer 29.00±3.00f28.33±0.58f22.33±2.73bcde 20.33±1.53bc 35.00±2.00g
Autumn 22.67±2.08bcde 24.33±2.73cde 23.67±1.15de 14.67±1.15a30.67±2.08f
Melittin -30.67±2.89fg 23.33±0.58d21.00±2.00bc 19.33±1.53b31.33±2.73fg
aResults are Means±SE of triplicates. Values within a column followed by different letters are significantly different by Tukey’s studentized
range test (α = 0.05); bMRSA; Methicillin-resistant Staphylococcus aureus; cAMP10: Ampicillin 10µg, CHL30: Chloramphenicol 30 µg, ERY15:
Erythromycin 15 µg, NA30: Nalidixic acid 30 µg, P10: Penicillin G (10 units), S10: Streptomycin10 µg, VA30: Vancomycin 30 µg. The resistance
for AMP10, P10, and S10 when inhibition zone (IZ) ≤ 11 mm; for CHL30 when IZ ≤ 12 mm, for ERY15, NA30, and VA30 when IZ ≤ 13 mm; dThe
inoculum concentration of honey, royal jelly, and propolis is 200 mg/ml; that of venom and melittin is 10 mg/ml.
Brazilian Journal of Biology, 2024, vol. 84, e2867316/15
Obeidat, M., Haddad, M.A. and Ghnamat, S.A.
activity against all antibiotic-resistant Gram-negative
bacteria tested in this study with the exception of the
clinical strain H. influenzae type b (Table 3). The honey
extract obtained from the three seasons, on the other
hand, inhibited the growth of H. influenza. The growth
of E. coli ATCC 8739 and K. pneumoniae ATCC 7700 was
significantly inhibited by honey extract prepared from
spring samples. The RJ extracts from spring, summer, and
autumn samples were found to produce similar ranges
of inhibition zones against the growth of each Gram-
positive bacterium, excluding S. pneumoniae, which was
resistant to RJ extracts (Table 2). In the same way, spring
and summer, as well as autumn extracts of RJ, also showed
similar patterns of inhibitory effects against the growth of
Gram-negative bacteria, including; K. pneumoniae ATCC
7700, P. vulgaris ATCC 33420, and H. influenza (Table 3).
Also, the growth of S. typhimurium ATCC 14028 and E. coli
ATCC 8739 was similarly inhibited via RJ that was collected
during spring and summertime. However, P. aeruginosa
ATCC 27253 and H. influenzae type b were found resistant
to RJ extracts. In comparison to the propolis extract
produced from autumn samples, the prepared extracts
from spring and summer samples significantly inhibited
the growth of S. pneumoniae ATCC 6305 and the clinical
strains of S. pneumoniae and S. aureus (Table 2). Moreover,
Table 3 demonstrated that there were no significant
differences observed between the sizes of the developed
zones of inhibition around S. aureus ATCC 95047 and MRSA
ATCC 95047, as well as around S. typhimurium ATCC 14028,
E. coli ATCC 8739, P. aeruginosa ATCC 27253, and P. vulgaris
ATCC 33420 (Gram-negative bacteria) after treatment
with propolis extracts that were collected from the three
seasons. While the growth of K. pneumoniae ATCC 7700 and
H. influenzae was significantly inhibited via propolis
extracts from springtime. Nevertheless, H. influenzae
type b was resistant to propolis extracts. Despite that
S. pneumoniae was resistant to honey and RJ extracts and
was moderately inhibited by propolis extracts (inhibition
zone diameters were 13.67±1.15 mm for the spring sample
and 10.67±2.08 mm for the summer sample), BV extracts
from different seasons and the standard MEL exhibited
effective bactericidal activity toward S. pneumoniae
(Table 2). Although H. influenzae type b was resistant
to honey, RJ, and propolis extracts, seasonally collected
BV extracts and MEL showed various inhibitory effects
against H. influenzae type b (Table 3). Interestingly, it was
observed that the BV extract prepared from the spring
sample significantly inhibited the growth of S. aureus
ATCC 25923, MRSA ATCC 95047, S. pneumoniae, S. aureus
(Table 2), P. aeruginosa ATCC 27253, P. vulgaris ATCC 33420,
and H. influenzae type b (Table 3) as compared to that of
standard MEL. In addition, the growth of S. pneumoniae
ATCC 6305, S. typhimurium ATCC 14028, E. coli ATCC 8739,
K. pneumoniae ATCC 7700, and H. influenzae was equally
inhibited by the extracts of BV (spring sample) and MEL.
It was noticed that the antibacterial effects of MEL are
similar to those produced by BV extracts of summer
samples toward Gram-positive and Gram-negative bacteria
except S. aureus ATCC 25923 and K. pneumoniae ATCC
7700, respectively.
The antifungal activity of natural bee products was
also tested in the current study at 200 mg/ml (honey, RJ,
and propolis) and 10 mg/ml (BV and MEL) concentrations
(Table 4). In comparison to honey extract from the autumn
season, it was found that honey extracts prepared from
spring and summer samples displayed significant antifungal
activity toward A. brasiliensis ATCC 16404, C. albicans ATCC
10231, and the clinical strain A. brasiliensis. The growth
of C. albicans was equally inhibited by all honey extracts.
On the other hand, RJ extracts from various seasons showed
similar inhibitory patterns against each fungus investigated
in this study. A similar result was also achieved for propolis
extracts that were prepared from samples collected from
different seasons, but the growth of C. albicans ATCC
10231 was significantly inhibited by propolis extracts
obtained from spring and summer samples as compared
to autumn samples. For BV extracts, it was noticed that
BV extracts produced from spring and summer samples in
comparison to autumn samples were significantly inhibited
by the clinical fungal strains A. brasiliensis and C. albicans, as
well as the reference strains A. brasiliensis ATCC 16404 and
C. albicans ATCC 10231. The growth of C. albicans ATCC
10231 was significantly inhibited (29.67±2.08 mm) via the
BV extract from the spring sample. Table 4 demonstrated
that MEL exhibited significant antifungal effects, which are
similar to those of the BV extract prepared from the spring
sample, against C. albicans ATCC 10231, A. brasiliensis, and
C. albicans. The growth of A. brasiliensis ATCC 16404 was
significantly inhibited by the spring sample of BV extract
in comparison to MEL.
In general, natural bee products collected in springtime
exhibited significant antimicrobial activity. Therefore, the
results of the antimicrobial activities of honey, RJ, propolis, BV,
and MEL samples collected during spring were confirmed by
estimating their MIC, MBC, and MFG values (Table 5). It was
indicated that honey extract had a lower MIC value against
S. aureus ATCC 25923 (16 mg/ml) than other Gram-positive
and Gram-negative bacteria. The extract of RJ had the lowest
MIC value for S. aureus ATCC 25923 and for the clinical
strain S. aureus, which had the lowest MBC value (8 mg/ml).
Furthermore, the MIC and MBC values of propolis extract were
the lowest (1 mg/ml for MIC and 2mg/ml for MBC) against
S. aureus. On the other hand, it was observed that the MIC
values of propolis extract were lower than those of RJ extract
for both Gram-positive and Gram-negative bacteria, and the
MIC values of RJ extract were lower than those of the honey
extract against Gram-positive bacteria (Table 5). However, the
MIC and MBC values of honey and RJ extracts against Gram-
negative bacteria were approximately the same. According
to MIC, MBC, and MFC values (Table 5), it was observed that
BV and its major component MEL have lower MIC and MBC
values than other bee products (honey, RJ, and propolis),
suggesting that BV and MEL are more effective as antibacterial
agents. Table 5 showed that BV extracts and MEL against the
clinical strain S. pneumonia had the lowest MIC (16 and 32 µg/
ml for BV and MEL, respectively) and MBC (32 µg/ml for BV
and 46 µg/ml for MEL) values. Moreover, MIC values of BV
extract were lower than those of MEL against Gram-positive
bacteria except for S. aureus, which had an equal MIC and a
lower MBC. The MIC values of BV extract on Gram-negative
bacteria (S. typhimurium ATCC 14028, K. pneumoniae ATCC
Brazilian Journal of Biology, 2024, vol. 84, e286731 7/15
Antimicrobial activities of bee products
Table 3. Antibacterial activity of honey, royal jelly, propolis, venom, and melittin that are seasonally collected from Apis mellifera bees against pathogenic Gram-negative bacteria.
Inhibition Zone (mm)a
S
.
typhimurium
ATCC 14028
E
.
coli
ATCC 8739
P
.
aeruginosa
ATCC 27253
K
.
pneumoniae
ATCC 7700
P
.
vulgaris
ATCC
33420
H. influenzae H. influenzae
type b
Antibiotic
Resistanceb
AMP10, NA30,
P10, VA30
AMP10, ERY15,
VA30
AMP10, CHL30,
ERY15, P10, S10
AMP10, P10, VA30 AMP10, P10 AMP10, CHL30,
ERY15, P10, S10
AMP10, CHL30,
ERY15, NA30, P10,
S10, VA30
Bee productcSeason
Honey Spring 16.67±2.89bcd 20.67±2.52def 17.00±1.00b23.67±1.15f19.33±1.53cdef 24.67±2.08f0a
Summer 17.33±1.15cd 13.33±2.08b14.67±2.73b20.33±1.89de 17.33±0.58c22.00±2.00ef 0a
Autumn 13.00±2.00b14.67±2.89b12.33±1.53b18.00±1.00cd 17.33±1.53cd 18.33±1.15cd 0a
Royal Jelly Spring 14.33±0.58e10.67±2.31bcd 0a22.33±3.51fg 16.00±3.00def 21.33±1.53g0a
Summer 12.33±2.08cde 9.67±2.52bc 0a20.33±3.51fg 16.67±2.73e19.33±1.89fg 0a
Autumn 8.33±1.53b0a0a21.67±1.15g14.00±2.00de 21.67±2.08g0a
Propolis Spring 14.67±2.89bcdef 16.67±1.15def 16.00±1.00cdef 21.67±1.15h15.33±1.53bcdef 21.33±1.89h0a
Summer 15.33±1.15cdef 18.67±2.52fgh 12.67±2.73bc 17.33±1.89def 13.33±0.58b17.67±1.15fg 0a
Autumn 16.00±1.00cdef 15.33±1.53bcdef 12.67±2.89bcdef 16.33±1.89cdef 14.00±2.00bcde 18.67±2.89efgh 0a
Venom Spring 25.67±2.08fgh 27.67±2.52ghij 26.33±1.53gh 32.67±3.06jk 29.67±1.15jk 33.67±1.15k18.33±3.06bcde
Summer 22.33±1.53ef 23.67±2.73efg 23.67±2.89efg 22.67±2.89defg 30.33±3.06hijk 30.00±3.00hijk 14.33±1.89ab
Autumn 19.00±1.00c17.33±1.53b20.00±3.00cde 19.67±3.51bcde 23.00±3.00defgh 27.33±0.58hi 13.67±1.15a
Melittin 21.00±1.00defgh 24.67±1.15fg 23.67±1.53f30.67±1.15jk 24.67±3.21efgh 29.33±0.58jk 14.33±0.58a
aResults are Means±SE of triplicates. Values within a column followed by different letters are significantly different by Tukey’s studentized range test (α = 0.05); bAMP10: Ampicillin 10µg, CHL30: Chloramphenicol 30
µg, ERY15: Erythromycin 15 µg, NA30: Nalidixic acid 30 µg, P10: Penicillin G (10 units), S10: Streptomycin10 µg, VA30: Vancomycin 30 µg. The resistance for AMP10, P10, and S10 when inhibition zone (IZ) ≤ 11 mm;
for CHL30 when IZ ≤ 12 mm, for ERY15, NA30, and VA30 when IZ ≤ 13 mm; cThe inoculum concentration of honey, royal jelly, and propolis is 200 mg/ml; that of venom and melittin is 10 mg/ml.
Brazilian Journal of Biology, 2024, vol. 84, e2867318/15
Obeidat, M., Haddad, M.A. and Ghnamat, S.A.
7700, P. vulgaris ATCC 33420, and H. influenzae type b) were
lower than those of MEL. The MBC values of honey and RJ
extracts on S. typhimurium ATCC 14028 and K. pneumoniae
ATCC 7700 were high. Table 6 shows the MIC and MFC values
for natural bee products collected in the springtime against
fungi. Ethanolic extracts of honey, RJ, and propolis produced
64-128, 32-64, and 2-4 mg/ml MIC values toward tested fungi
(A. brasiliensis ATCC 16404, C. albicans ATCC 10231, and the
clinical strains of A. brasiliensis and C. albicans); the lowest
MIC (2 mg/ml) was generated on C. albicans. The MIC and
MFC values of propolis were lower than those produced
by RJ extract which had lower MIC and MFC values than
honey extract (fungicidal effects of honey and RJ extracts on
C. albicans ATCC 10231 had equal MFC values). Furthermore, BV
extracts and MEL had lower MIC and MFC values than honey,
RJ, and propolis extracts (Table 6). In addition, BV extracts
had lower MIC values than MEL against A. brasiliensis ATCC
16404, C. albicans ATCC 10231 (MIC of 64 µg/ml is the lowest
value), A. brasiliensis, and C. albicans. Likewise, BV extracts
had lower MFC values than MEL on all tested fungi. The MFC
value produced by BV on A. brasiliensis ATCC 16404 was the
lowest (128 µg/ml) and the MFC value produced by MEL
on C. albicans ATCC 10231 was the highest (2.048 mg/ml).
4. Discussion
The natural bee products examined in this study (honey,
propolis, RJ, BV, and MEL) exhibited promising antibacterial
and antifungal properties against antibiotic-resistant
bacteria and fungi. Therefore, they can be considered a
good candidate to generate effective protection against
bacterial and fungal infections that have developed
resistance to standard antibiotics.
Ethanol was selected as an organic solvent in the current
study to extract bee products because it had previously
been proven (Elswaby et al., 2022) that ethanol provided
the greatest antioxidant potential. Moreover, Elswaby et al.
(2022) reported that the antimicrobial activity of ethanolic
extracts of bee products (propolis and BV) was more
efficient than that of other solvents such as chloroform
and water. Ethanolic extraction of propolis was found to
remove wax and organic debris, and the propolis tincture
(balsam) thus obtained contains the bulk of propolis
bioactive constituents (Tsibranska et al., 2011). Also, 70%
ethanol coupled with agitation was found to be the best
extracting process for some bee products (Lawag et al.,
2021). In this study, ethanol was also selected as an organic
solvent for the BV extraction process because it does not
affect the MEL content or its stability in BV (Lee et al., 2018).
This study found that extracts of natural bee products
collected in spring, summer, and autumn had varying
inhibitory effects on antibiotic-resistant Gram-positive
bacteria (three reference strains; S. pneumoniae ATCC
6305, S. aureus ATCC 25923, and MRSA ATCC 95047, and
two clinical strains; S. pneumoniae and S. aureus), Gram-
negative bacteria (five reference strains; S. typhimurium
ATCC 14028, E. coli ATCC 8739, P. aeruginosa ATCC 27253,
K. pneumoniae ATCC 7700, P. vulgaris ATCC 33420, and
Table 4. Antifungal activity of honey, royal jelly, propolis, venom, and melittin that are seasonally collected from Apis mellifera bees
against pathogenic fungi.
Inhibition Zone (mm)a
A
.
brasiliensis
ATCC 16404
C
.
albicans
ATCC 10231
A
.
brasiliensis C
.
albicans
Antibiotic ResistancebCYX250, NYS10 NYS10 CYX250, NYS10 CYX250, NYS10
Bee productcSeason
Honey Spring 22.67±2.08efg 21.67±2.89def 26.33±1.53g22.67±2.89defg
Summer 19.33±1.53cde 16.67±2.52bcd 22.33±1.15fg 18.67±2.31cde
Autumn 13.00±2.00ab 11.67±2.08a16.67±1.15c16.33±3.51bcd
Royal Jelly Spring 12.33±1.73ab 14.67±2.31b13.67±2.52b12.67±1.15b
Summer 12.67±2.08ab 14.67±2.89b10.33±0.58a14.33±1.73b
Autumn 10.33±0.58a12.67±1.15b11.33±1.89ab 11.33±1.53b
Propolis Spring 17.33±1.53d15.67±2.52bcd 16.00±3.00abcd 14.67±2.89abcd
Summer 14.67±2.09abcd 12.67±2.08ab 16.67±2.73bcd 13.67±2.31abcd
Autumn 15.67±2.08bcd 11.33±1.73a13.00±2.00ab 10.33±3.51ab
Venom Spring 31.33±1.53e29.67±2.08de 30.67±2.31de 27.33±1.53cd
Summer 29.00±1.00de 26.33±0.58c27.67±2.52cde 25.33±1.89bc
Autumn 26.33±0.58c23.00±2.00ab 22.33±0.58a22.67±2.08ab
Melittin 27.33±1.53cd 27.33±1.73cd 28.67±2.31cde 28.33±0.58d
aResults are Means±SE of triplicates. Values within a column followed by different letters are significantly different by Tukey’s studentized
range test (α = 0.05). bCYX250: Cycloheximide 250 µg and NYS10: Nystatin 10 µg. The resistance for CYX250 and NYS10 when IZ ≤ 8 mm. cThe
inoculum concentration of honey, royal jelly, and propolis is 200 mg/ml; venom and melittin is 10 mg/ml.
Brazilian Journal of Biology, 2024, vol. 84, e286731 9/15
Antimicrobial activities of bee products
Table 5. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of bee products collected in the spring season.
Bee Product
MIC (MBC) in mg/ml
Gram-positive bacteria Gram-negative bacteria
1a2 3 4 5 6 7 8 9 10 11 12
Honey 64 (64) 16 (32) 64 (64) - 32 (32) 128 (˃128) 64 (128) - 128 (˃128) 64 (128) 64 (128) -
Royal Jelly 32 (32) 8 (16) 16 (32) - 8 (16) 128 (˃128) 32 (128) - 128 (˃128) 128 (128) 64 (64) -
Propolis 4 (8) 2 (4) 4 (4) 8 (8) 1 (2) 32 (64) 32 (64) 64 (128) 6 4 (64) 32 (128) 16 (64) -
Venom 0.064 (0.128) 0.032 (0.064) 0.128 (0.256) 0.016 (0.032) 0.064 (0.064) 1.024 (2.048) 0.512 (0.512) 1.024 (2.048) 0.512 (1.024) 0.256 (0.512) 0.256 (0.256) 0.256 (0.512)
Melittin 0.128 (0.128) 0.128 (0.256) 0.256 (0.256) 0.032 (0.0 64) 0.06 4 (0.128) 2.048 (˃2.048) 0.512 (1.024) 1.024 (2.048) 1.024 (1.024) 0.512 (0.512) 0.256 (0.512) 0.512 (1.024)
a1; S. pneumoniae ATCC 6305, 2; S. aureus ATCC 25923, 3; MRSA ATCC 95047, 4; S. pneumoniae, 5; S. aureus; 6; S. typhimurium ATCC 14028, 7; E. coli ATCC 25922, 8; P. aeruginosa ATCC 27253, 9; K. pneumoniae ATCC
7700, 10; P. vulgaris ATCC 33420, 11; H. influenzae, 12; H. influenzae type b.
Brazilian Journal of Biology, 2024, vol. 84, e28673110/15
Obeidat, M., Haddad, M.A. and Ghnamat, S.A.
two clinical strains; H. influenzae and H. influenzae type
b), and fungi (two reference strains; A. brasiliensis ATCC
16404, C. albicans ATCC 10231, and two clinical strains;
A. brasiliensis, C. albicans). These results were in agreement
with several previous studies that reported the effectiveness
of natural bee products as antimicrobial agents. Honey
extracts showed antibacterial and antifungal activities
against all microorganisms tested in this study except the
clinical strains of S. pneumonia and H. influenzae type b. This
is consistent with preceding reports that documented the
effectiveness of honey as an antimicrobial agent against
S. aureus ATCC 25923 (Maželien
ė
et al., 2022), MRSA
(Jantakee and Tragoolpua, 2015), and S. aureus (Jantakee
and Tragoolpua, 2015; Morroni et al., 2018; Moselhy et al.,
2013; Srećković et al., 2019). Srećković et al. (2019) showed
that cultures of S. typhimurium, E. coli, P. aeruginosa, and
K. pneumoniae were inhibited by honey extracts. Similar
results were also obtained by Wadi (2022), who reported
the susceptibility of P. vulgaris to honey extracts in addition
to S. aureus, MRSA, E. coli, P. aeruginosa, and K. pneumonia.
Al-Waili (2004) and Huttunen et al. (2013) reported
that the growth of H. influenzae and S. pneumoniae was
inhibited via honey extracts, but their growth was not
inhibited in this study. The antifungal activity results were
in agreement with Srećković et al. (2019), who showed that
the growth of A. brasiliensis and C. albicans was inhibited
by honey extracts.
The results of the antimicrobial activity of RJ obtained
in this work were in agreement with other studies and
supported by them. Maželien
ė
et al. (2022) and Al-
Abbadi (2019) demonstrated that RJ extracts displayed
antibacterial activity against S. aureus ATCC 25923.
Recently, Uthaibutra et al. (2023) supported these results
and reported that RJ extracts were able to inhibit S. aureus
and MRSA. A good antibacterial effect of RJ fatty acids
against S. pneumoniae has been reported (Nascimento et al.,
2015). However, this is inconsistent with the result of this
study; the growth of S. pneumoniae was not inhibited
by RJ extracts. The resistance of S. pneumoniae could be
attributed to its multi-drug resistance to AMP10, CHL30,
ERY15, NA30, P10, S10, and VA30. The effectiveness of
RJ extracts in the inhibition of Gram-negative bacteria,
S. typhimurium ATCC 14028, E. coli ATCC 8739, P. aeruginosa
ATCC 27253, and P. vulgaris ATCC 33420, was supported by
Al-Abbadi (2019). In contrast, Al-Abbadi (2019) claimed
that P. aeruginosa ATCC 27253 growth was suppressed
by ethanolic extracts of RJ, which is in disagreement
with the current investigation. Moreover, RJ extracts
inhibited the growth of K. pneumoniae (Al-Abbadi, 2019;
Maželienė et al., 2022), which is in agreement with the
results of this study. This study tested the bactericidal
activity of RJ against H. influenzae strains (Table 3) and it
was found that H. influenzae type b, which had resistance
toward seven antibiotics examined in this study (AMP10,
CHL30, ERY15, NA30, P10, S10, VA30), was also resistant to
ethanolic extracts of RJ. Consistent with antifungal results,
Srećković et al. (2019) reported that both A. brasiliensis
and C. albicans were inhibited via RJ.
Ethanolic extracts of propolis exhibited a wide
spectrum of antibacterial activity against S. aureus, MRSA,
S. typhimurium, E. coli, P. aeruginosa, K. pneumoniae, and
P. vulgaris, this result is in agreement with Al-Abbadi et al.
(2015). The bactericidal activity of propolis toward
H. influenzae was reported in the current study and
confirmed by the previous study of Drago et al. (2007), who
observed that Actichelated® propolis and hydroalcoholic
propolis had potent inhibitory effects on the growth of
H. influenzae. However, the growth of H. influenzae type b
was not inhibited, which might be due to its wide spectrum
of antibiotic resistance. On the other hand, the results
of this study illustrated for the first time that ethanolic
extracts of propolis inhibited the growth of S. pneumoniae.
Like honey and RJ, propolis exhibited antifungal activity
against A. brasiliensis and C. albicans, which is in agreement
with Al-Abbadi et al. (2015) and Kalogeropoulos et al.
(2009), who demonstrated that propolis had antifungal
activity against A. brasiliensis and C. albicans, respectively.
In terms of antimicrobial activities of BV ethanolic
extracts, it was found that BV and its major component
MEL had both antibacterial and antifungal inhibitory
effects on the bacteria and fungi tested in this study. This
is in agreement with previous reports (Han et al., 2016;
Kim et al., 2006) that demonstrated extracts of BV had
antibacterial activity on the growth of MRSA and S. aureus,
and there are several reports (Askari et al., 2021; Choi et al.,
2015; Ko et al., 2020) supporting the effectiveness of MEL
against MRSA. In addition, inhibition of S. aureus growth
by MEL was previously described (Choi et al., 2015;
Ko et al., 2020; Yokota et al., 2001). Choi et al. (2015) also
reported that BV inhibits the growth of S. pneumoniae.
Several previous reports documented the bactericidal
activity of BV extracts against Gram-negative bacteria,
including S. typhimurium, E. coli (Elswaby et al., 2022;
Zolfagharian et al., 2016), P. aeruginosa (Frangieh et al.,
Table 6. Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of bee products collected in the
spring season.
Bee product
MIC (MFC) in mg/ml
A
.
brasiliensis
ATCC 16404
C
.
albicans
ATCC 10231
A
.
brasiliensis C
.
albicans
Honey 128(128) 64(128) 128(128) 128(128)
Royal Jelly 64(64) 32(128) 32(64) 64(64)
Propolis 4(16) 4(8) 4(4) 2(4)
Venom 0.128(0.128) 0.256(0.256) 0.128(0.256) 0.256(0.512)
Melittin 0.256(0.512) 0.256(2.048) 0.128(1.024) 0.256(1.024)
Brazilian Journal of Biology, 2024, vol. 84, e286731 11/15
Antimicrobial activities of bee products
2019), and K. pneumoniae (Issam et al., 2015; Jamasbi et al.,
2018), which are in agreement with the results of this study.
Consistent with the findings of Jamasbi et al. (2018) and
Ko et al. (2020), the results showed that MEL inhibited
the growth of E. coli, P. aeruginosa, and K. pneumoniae.
Baker et al. (1997) reported that S. typhi, which is a species
related to S. typhimurium, was sensitive to MEL. The results
of this study showed for the first time that the growth of
P. vulgaris ATCC 33420, and the clinical strains H. influenzae
and H. influenzae type b were inhibited by BV extracts and
MEL. Ethanolic extracts of BV and MEL exhibited antifungal
activity against C. albicans, and this is in agreement with
Elswaby et al. (2022), who reported that C. albicans was
sensitive to the ethanolic extracts of BV, and Lee and
Lee (2010), who showed that MEL produced inhibition
against C. albicans. The inhibitory effects of BV and MEL
on A. brasiliensis that were observed in this study were
in agreement with Elshehaby (2022), who showed that
BV significantly inhibited the growth of related species,
A. niger and A. flavus at 300 and 600 µg/ml, respectively.
Moreover, Lee and Lee (2010) demonstrated that MEL
inhibited the growth of A. fumigatus, a species related to
A. brasiliensis. Furthermore, Memariani and Memariani
(2020) reported that MEL had antifungal activity against
Aspergillus.
It is important to notice that the constituents and
properties of bee natural products vary from season to
season as a result of changes in bee metabolic activity,
climate, botanical sources, and nectars. Consequently, the
antimicrobial properties of these products will differ greatly.
This was confirmed in this study by evaluating TPC and
TFC in honey, RJ, and propolis; calculating the percentage
of MEL content in the BV; and comparing the bactericidal
and fungicidal activities of bee products collected from
different seasons (spring, summer, and autumn). It was
found that TPC and TFC were higher in spring samples of
honey, RJ, and propolis (Table 1) as compared to summer
and autumn samples. As well, analysis of the BV constituent
of MEL showed that the highest percentage of MEL content
was in BV samples collected in the spring season and
comprised 25.58% to 33.92% of dry venom. In general, bee
products collected during spring had higher antibacterial
and antifungal activities. This high antimicrobial potency of
spring samples of honey, RJ, and propolis against bacteria
and fungi could be attributed to their higher contents of
phenolic compounds and flavonoids compared to summer
and autumn samples. Compared to summer and autumn
samples of BV, the spring samples showed the greatest
antimicrobial potency as a result of their higher content
of MEL peptide. Therefore, it is generally accepted that
bee products harvested during the spring season were the
richest in various constituents, which in turn produced the
greatest antimicrobial potency. Several reports supported
the antimicrobial results of this study regarding the spring
season: Malagnini et al. (2022) reported that pollen
diversity and protein content in honey are affected by
season and landscape composition heterogeneity, and
are highest in spring (flowering season); Hussain et al.
(2020) demonstrated that RJ production is affected by
seasons, with spring having the highest percentage
of RJ per colony; Kekeço
ğ
lu et al. (2021) showed that
the chemical composition of propolis such as phenolic
compounds and its antioxidant capacity are affected by
season, and they are the richest in propolis collected in
the spring season; Huang et al. (2020) observed variable
percentages of MEL content in BV throughout the year and
hypothesized that MEL content is affected by the season
of BV collection, with BV collected in the spring season
containing a higher percentage of MEL; Lee et al. (2018)
reported that seasonal fluctuations in BV composition
may occur due to change in flowers and fruits, and thus
bee feeding and MEL production vary with the seasons.
Based on MIC and MBC values produced by samples
collected in the spring, it was found that Gram-positive
bacteria are more susceptible to all bee products (honey, RJ,
propolis, BV, and MEL) than Gram-negative bacteria. This
finding is in agreement with several previous studies that
demonstrated the higher susceptibility of Gram-positive
bacteria to natural bee products than Gram-negative
bacteria. Zainol et al. (2013) and Tuksitha et al. (2018)
showed that honey exhibited more active antibacterial
activity against Gram-positive bacteria than Gram-
negative bacteria. Fujiwara et al. (1990) elucidated that
low concentrations of royalisin protein found in RJ had
potent antibacterial activity against Gram-positive bacteria
but not against Gram-negative bacteria. Moreover, it was
reported that (Khosla et al., 2020) Gram-positive bacteria
were more sensitive to the major RJ proteins, including
royalisin, jellenies, and enzymes such as GOx, than Gram-
negative bacteria, but jellenies are also effective against
Gram-negative bacteria and yeasts. Many previous studies
(Drago et al., 2000; Mirzoeva et al., 1997) illustrated that
the antibacterial effect of propolis on Gram-positive
bacteria was greater than that on Gram-negative bacteria.
Zolfagharian et al. (2016) reported that BV and its major
component MEL protein were more effective against
Gram-positive bacteria than against Gram-negative
bacteria. It was also reported that MEL was more active
against Gram-positive bacteria (Galdiero et al., 2019;
Nevalainen et al., 2008). The highest antibacterial activity
against Gram-positive bacteria may be due to the acidity
of honey (pH ~3.7), RJ (pH ~3.3), propolis (pH ~5.0), and BV
(pH ~4.9), enzymes content such as GOx found in honey
and RJ, as well as flavonoids and phenolic contents in
honey, RJ, and propolis (Table 1). Furthermore, this potent
antibacterial activity could be attributed to the differences
in the cell wall and membrane structure of Gram-positive
and Gram-negative bacteria. Consequently, bee products
or some of their constituents can easily penetrate the
thick peptidoglycan layer of the cell wall of Gram-positive
bacteria and reach the cell membrane, compared to Gram-
negative bacteria that are less susceptible to various bee
products due to the presence of the lipopolysaccharide
layer in their cell walls. Ceremuga et al. (2020) documented
that positively charged MEL has a cytolytic effect that
disrupts cell membranes lipids and has antibacterial
properties. Normally, MEL is coiled and bound to the cell
membrane, which is based on its ability to conform pores
to biological membranes. The hydrophobic section and its
positive load attract MEL to the anion lipid membranes,
which then insert MEL into the lipid membrane through
hydrophobic interactions.
Brazilian Journal of Biology, 2024, vol. 84, e28673112/15
Obeidat, M., Haddad, M.A. and Ghnamat, S.A.
Furthermore, the results of antimicrobial activity
of spring samples of honey, RJ, and propolis against
Gram-positive bacteria and fungi showed that propolis
had lower MIC values than RJ and that RJ had lower
MIC values than honey. This order of MIC values can be
attributed to the great variation in TPC and TFC between
propolis, RJ, and honey (Table 1), where propolis had the
highest content of both TPC (168.81±6.19 mg GAE/g) and
TFC (84.94±2.64 mg QE/g), but honey had the lowest
content (0.64±0.02 mg GAE/g and 0.048±0.002 mg QE/g
for TPC and TFC, respectively). To our knowledge, no
previous study has compared the MIC values for honey,
RJ, and propolis.
It was revealed that BV and its major component MEL
significantly inhibited the growth of bacteria and fungi
compared to other bee products; honey, RJ, and propolis.
This was confirmed by the differences in MIC, MBC, and
MFC values that were measured in mg/ml for extracts of
bee products. This difference in antimicrobial properties
has been attributed to different bioactive compounds.
Certainly, phenolics and flavonoids are present in honey,
RJ, and propolis, as well as GOx in honey and RJ, in addition
to other components such as proteins and fatty acids,
and MEL is the main component in BV (Bankova, 2005;
Bílikova et al., 2015; Ceremuga et al., 2020; Cooke et al.,
2015; Melliou and Chinou, 2005; Yupanqui Mieles et al.,
2022). This distinct composition of BV and other bee
products (honey, RJ, and propolis) might explain the
lower MIC, MBC, or MFC values of BV extracts of the same
geographical and botanical origins.
5. Conclusion
This study demonstrates that the season affects the
constituents of the bee products and, consequently, affects
their antimicrobial activity. As a result, it was found that
TPC and TFC were the highest in spring samples of propolis,
followed by RJ and honey, in that order, and the highest
MEL content in BV was also found in the spring sample.
In accordance with this, the antimicrobial activity of bee
products collected during the spring season was the most
potent. Based on the MIC values produced by the spring
samples, it was suggested that antimicrobial activity can be
sorted in descending order into BV, MEL, propolis, RJ, and
honey. No previous study established this sequential order
of antimicrobial activity for bee products. The abundance
of MEL in BV and consecutive decreases in TPC and TFC
in propolis, RJ, and honey were proposed to reflect this
sequential arrangement of antimicrobial activity. The results
indicate that Gram-positive bacteria are more susceptible
to bee products than Gram-negative bacteria, possibly due
to differences in the cell wall and membrane structure,
acidity of bee products, enzymes in honey and RJ, phenolic
and flavonoid content in honey, RJ, and propolis, and the
cytolytic effect of MEL found in BV. In conclusion, the
findings of the current study indicate that bee products
are a good candidate for providing effective antimicrobial
activities against antibiotic-resistant bacteria and fungi,
as well as a promising alternative to current antibiotics
for disease treatment.
Acknowledgements
This study was kindly supported by the Deanship
of Scientific Research and Innovation, Al-Balqa Applied
University (Grant number: 497/2020/2021).
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