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

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

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-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 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% Na₂CO₃ 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 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.

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.

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).

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	TPC a (mg GAE/g)	TFC b (mg QE/g)	Melittin%
Honey	Spring	16.53±2.29c	3.75±0.02b	0.64±0.02b	0.048±0.002b	-
	Summer	16.49±1.87c	3.71±0.11b	0.57±0.03a	0.041±0.004a	-
	Autumn	18.96±3.17c	3.73±0.04b	0.50±0.06a	0.035±0.010a	-
Royal Jelly	Spring	67.50±3.78d	3.32±0.06a	29.30±1.54d	2.29±0.12d	-
	Summer	66.35±2.01d	3.29±0.03a	25.34±0.67c	2.21±0.05d	-
	Autumn	66.30±2.67d	3.33±0.04a	23.48±1.33c	1.98±0.11c	-
Propolis	Spring	-	5.13±0.07e	168.81±6.19f	84.94±2.64g	-
	Summer	-	5.07±0.04de	150.11±11.17e	65.09±5.77f	-
	Autumn	-	5.09±0.02e	148.39±9.23e	54.36±3.83e	-
Venom	Spring	4.74±0.67a	4.93±0.12cd	-	-	29.75±4.17c
	Summer	6.89±1.33b	4.87±0.09c	-	-	14.50±4.03a
	Autumn	14.69±1.78c	5.01±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).
• ^a
  TPC, Total phenolic content; GAE, Gallic acid equivalents;
• ^b
  TFC, Total flavonoid content; QE, Quercetin equivalents.

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 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.

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	MRSA b ATCC 95047	S. pneumoniae	S. aureus
Antibiotic Resistance c		AMP10, P10, VA30	AMP10, ERY15, P10, VA30	AMP10, P10	AMP10, CHL30, ERY15, NA30, P10, S10, VA30	AMP10, ERY15, P10, S10, VA30
Bee product d	Season
Honey	Spring	23.67±2.08e	14.00±2.00c	18.67±1.15d	0a	28.33±2.73e
	Summer	15.33±0.58c	17.67±1.15d	13.67±1.53c	0a	26.67±2.89e
	Autumn	10.33±0.58b	13.33±2.73bc	9.00±2.00b	0a	18.33±0.58d
Royal Jelly	Spring	12.67±2.89bc	12.00±1.00bc	13.67±1.15bc	0a	21.33±0.58d
	Summer	11.33±1.53bc	11.67±2.89bc	10.67±2.08bc	0a	21.00±2.00d
	Autumn	8.67±3.06b	10.33±2.73bc	0a	0a	20.33±2.08d
Propolis	Spring	16.33±0.58e	12.67±1.53c	17.67±2.08e	9.33±1.53b	22.67±2.89f
	Summer	15.33±2.73cde	11.33±2.73bcd	15.67±1.15de	7.67±2.08b	21.00±1.00f
	Autumn	12.33±2.73bcd	12.33±2.73bcd	15.33±0.58de	0a	18.33±2.73e
Venom	Spring	32.67±3.06g	27.67±1.15f	25.67±1.15e	23.67±2.08cde	38.33±0.58h
	Summer	29.00±3.00f	28.33±0.58f	22.33±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±1.15a	30.67±2.08f
Melittin	-	30.67±2.89fg	23.33±0.58d	21.00±2.00bc	19.33±1.53b	31.33±2.73fg

• ^a
  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);
• ^b
  MRSA; Methicillin-resistant Staphylococcus aureus;
• ^c
  AMP10: 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;
• ^d
  The inoculum concentration of honey, royal jelly, and propolis is 200 mg/ml; that of venom and melittin is 10 mg/ml.

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 Resistance b		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 product c	Season
Honey	Spring	16.67±2.89bcd	20.67±2.52def	17.00±1.00b	23.67±1.15f	19.33±1.53cdef	24.67±2.08f	0a
	Summer	17.33±1.15cd	13.33±2.08b	14.67±2.73b	20.33±1.89de	17.33±0.58c	22.00±2.00ef	0a
	Autumn	13.00±2.00b	14.67±2.89b	12.33±1.53b	18.00±1.00cd	17.33±1.53cd	18.33±1.15cd	0a
Royal Jelly	Spring	14.33±0.58e	10.67±2.31bcd	0a	22.33±3.51fg	16.00±3.00def	21.33±1.53g	0a
	Summer	12.33±2.08cde	9.67±2.52bc	0a	20.33±3.51fg	16.67±2.73e	19.33±1.89fg	0a
	Autumn	8.33±1.53b	0a	0a	21.67±1.15g	14.00±2.00de	21.67±2.08g	0a
Propolis	Spring	14.67±2.89bcdef	16.67±1.15def	16.00±1.00cdef	21.67±1.15h	15.33±1.53bcdef	21.33±1.89h	0a
	Summer	15.33±1.15cdef	18.67±2.52fgh	12.67±2.73bc	17.33±1.89def	13.33±0.58b	17.67±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±1.15k	18.33±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±1.00c	17.33±1.53b	20.00±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±1.53f	30.67±1.15jk	24.67±3.21efgh	29.33±0.58jk	14.33±0.58a

• ^a
  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);
• ^b
  AMP10: 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;
• ^c
  The inoculum concentration of honey, royal jelly, and propolis is 200 mg/ml; that of venom and melittin is 10 mg/ml.

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.

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 Resistance b		CYX250, NYS10	NYS10	CYX250, NYS10	CYX250, NYS10
Bee product c	Season
Honey	Spring	22.67±2.08efg	21.67±2.89def	26.33±1.53g	22.67±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±2.08a	16.67±1.15c	16.33±3.51bcd
Royal Jelly	Spring	12.33±1.73ab	14.67±2.31b	13.67±2.52b	12.67±1.15b
	Summer	12.67±2.08ab	14.67±2.89b	10.33±0.58a	14.33±1.73b
	Autumn	10.33±0.58a	12.67±1.15b	11.33±1.89ab	11.33±1.53b
Propolis	Spring	17.33±1.53d	15.67±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±1.73a	13.00±2.00ab	10.33±3.51ab
Venom	Spring	31.33±1.53e	29.67±2.08de	30.67±2.31de	27.33±1.53cd
	Summer	29.00±1.00de	26.33±0.58c	27.67±2.52cde	25.33±1.89bc
	Autumn	26.33±0.58c	23.00±2.00ab	22.33±0.58a	22.67±2.08ab
Melittin		27.33±1.53cd	27.33±1.73cd	28.67±2.31cde	28.33±0.58d

• ^a
  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).
• ^b
  CYX250: Cycloheximide 250 µg and NYS10: Nystatin 10 µg. The resistance for CYX250 and NYS10 when IZ ≤ 8 mm.
• ^c
  The inoculum concentration of honey, royal jelly, and propolis is 200 mg/ml; venom and melittin is 10 mg/ml.

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 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).

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
	1 a	2	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)	64 (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 (0.064)	0.064 (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)

• ^a
  1; 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.

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)

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 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 (Maželienė 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. Maželienė 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., 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; Kekeçoğlu 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.

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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Publication Dates

• Publication in this collection
  13 Dec 2024
• Date of issue
  2024

History

• Received
  19 May 2024
• Accepted
  18 Sept 2024