Preliminary Study on Potential Antimicrobial Activity of Honey from Egricayir Plateau, Turkey

Safety and authenticity are indispensable attributes of any honey. Antimicrobial activity (AMA) is attributed to some, and because of therapeutic effects these honeys are considered more valuable than regular ones. The AMA has emerged as a robust tool to add further value to honey as an indicator of therapeuticity, and determining it continues to gain significant consideration and mainly focuses on microbial effects, action mechanisms, clinical uses and honey sources (Combarros-Fuertes et al., 2019; Combarros-Fuertes et al., 2020a).

Approaches for determining the AMA of honeys and honey products have been extensively discussed. The phenol equivalence test has been largely adopted by the industry for the practical assessment and categorization of honey (Hossain et al., 2022). The test determines the AMA against Staphylococcus aureus ATCC 25923, due to the peroxide and non-peroxide components of honey and shortened as total activity (TA). It is a variant of the agar well diffusion method (Balouiri et al., 2016) and compares the inhibition zone formed by the honey sample and standard phenol solutions.

Honeys already have inherent basic AMA due to high sugar content, low water activity, acidic pH and bioactive compounds (Combarros-Fuertes et al., 2020b). However, besides the basic one, the therapeutic effect is mainly linked to the AMA coming from hydrogen peroxide as in 'peroxide honeys' or methylglyoxal (MGO) as in 'non-peroxide honeys' (Mandal & Mandal, 2011). Honeys with TA≥10, i.e. AMA of honey ≥ AMA of 10% phenol solution, are considered therapeutically beneficial (Carter et al., 2010), and the more potent the honey the higher the TA.

Turkey, a leader in pine honey production, possesses a diverse and rich indigenous flora of more than 10,000 endemic flowers suitable for beekeeping and blossom honey production (Arslan & Turhan, 2022). Such fertile land should have a considerable portfolio of honeys with therapeutical AMA. However, research on the AMA of Turkish honeys is scarce compared to its rich honey resources.

Some Turkish honeys were investigated for AMA (Cenet et al., 2015; Bayram et al., 2020; Kemal et al., 2023), which was reported through zone diameter, minimum inhibition concentration, etc. which are expressive for the academic community. Most honeys from other geographies were similarly studied for their AMA (Junie et al., 2016; Mduda et al., 2023; Stagos et al., 2018), and their outcomes were not comparable due to the use of different methodologies. The therapeutic potencies of honeys were not assessed and not categorized with the use of a standard method. Expressing AMA in terms of TA is regarded as more advantageous for both industry and academia.

Besides the vague rare evidence for AMA of Turkish honeys, some are colloquially alleged to have healing effects and are used folklorically, as in the case of the honey from the Egricayir Plateau, Mersin, Turkey. These anecdotes prompted this search to pursue supporting evidence for the honey from the Plateau. So, the objective of the current preliminary work is to determine the AMA of this rare honey on the scale of TA in order to assess and categorize its potential therapeutic potency with the use of the phenol equivalence test.

Honey Samples

Honey samples were collected from five hives in the main honey-foraging region of the Egricayir Plateau, Turkey, Mersin (Fig. 1). Hives were selected among the existing ones within the smallest imaginary rectangle circumambienting the terrain. One of the hives was almost in the geometrical center of the rectangle, and the others were accordingly on the lengthways through the center. Each hive was almost 1000 m away from the others (Fig. 1).

Three different honeycombs (HCs) in a hive were arbitrarily selected for sampling at a collection time (CT). Samples were collected in early July and late August 2023. Half of each HC was cut out with the use of a sterile knife, put into separate sterile containers and kept dark and cold till analysis. Honey draining by itself from the HCs into the container was strained with the use of a sterile house-type strainer into sterile bottles. In total, thirty (2×3×5) TA analyses were performed for a CT. Furthermore, 5 g of honey used for each TA analysis was mixed (total 150 g) and analyzed by an accredited external laboratory (MRL Food Control Laboratory, Mersin, Turkey) (Tab. 1) to present the average physicochemical parameters of the mixture.

Inoculation

AMA of phenol solutions and honey samples were tested against Staphylococcus aureus ATCC 25923 with the use of the agar well diffusion method to determine the TA according to the phenol equivalence test by Allen et al. (1991). S. aureus culture was obtained from the stock culture collection of the Microbiology Laboratory, Department of Food Engineering, University of Mersin.

Mueller Hinton broth medium (Merck, Darmstadt, Germany) was prepared according to the manufacturer's instructions. It was autoclaved (15 min, 121°C), and allowed to cool to 50°C. S. aureus from the stock culture was inoculated into the broth with the use of a sterile loop. The S. aureus was grown in the broth for 18–24 h at 37°C and then adjusted to McFarland standard 0.5 (~10⁸ cfu/ml) with the use of a spectrometer (Agilent, Cary 60 UV-Vis, Santa Clara, CA, USA). Mueller Hinton agar medium was prepared, autoclaved (15 min, 121°C), and kept at 50°C for 30 minutes. A 100 µl broth was seeded into 150 ml agar and mixed gently through swirling, then poured into sterile Petri dishes (20 ml). Petri dishes were left to cool to room temperature and kept at 4°C overnight. The thickness of the agar was almost 2.40 mm. Two wells were bored in each Petri dish with the use of a sterile cork borer with 8 mm inner diameter.

Phenol Equivalence Test

A stock solution of 10% (w/v) phenol was prepared in a sterile 100 ml volumetric flask. Phenol solutions from 1 to 8% (w/v) were prepared with sterile pure water. Three Petri dishes were used for each phenol solution. One of the wells in a petri dish was delivered 100 μL sterile water as the negative control and the other well was delivered 100 μL phenol solution. Dishes were incubated at 37°C for 24 h. After the incubation, the inhibition zone diameter (ZD) formed was measured with the use of a vernier caliper at least twice on different sites. The squared means of ZD values versus phenol concentration were drawn to obtain the calibration curve (Fig. 2).

Each honey sample was diluted to one-fourth to obtain a 25% w/v honey solution. Two Petri dishes were used for each honey sample. Dishes were incubated at 37°C for 24 h. After the incubation, the ZD was measured at least twice. The squared mean of ZD values was determined. The squared values for the honey solutions were compared with those of the phenol solutions with the use of the calibration curve (Fig. 2). The corresponding readings were multiplied by the dilution factor 4 to obtain the TA.

Colony Forming Units

The number of colony-forming units (cfu) on agar gels was counted before and after the incubation for arbitrarily selected five honey samples from each run and for every phenol solution. Approximately 1 g agar was drawn from the designated zones on gels with the use of sterile tweezers, put into a sterile 10 ml volumetric flask, completed to 10 ml, and diluted with the peptone water down to 10⁻⁷. From the last dilution, 100 μL was homogeneously spread on Petri dishes. They were incubated at 37°C for 24 h and cfu were counted under a magnifying glass.

Statistical Analysis

The inferential and descriptive statistical analysis for ZD and TA values of honey samples was performed with IBM SPSS for Windows 25.0 (IBM, 2017). The inferential analysis was performed through ANOVA for p < 0.05.

Egricayir Plateau

The area within the 15 km radius of the coordinates on the 36° 58′ 25″ N and 33° 49′ 31″ E on the Taurus Mountains is recognized as the Egricayir Plateau, Mersin, Turkey. The honey named after the Plateau is foraged effectively over almost 14,000 acres of the landscape at an altitude of around 2000 meters. The vegetation is mostly prairie plants, and the main nectar sources are thyme, keven, cornflower, wild clover, thistle, heather, phacelia, chaste berry, and unknown plants. The nectar flow occurs between mid-May and August, and the honey harvested is polyfloral. The harvesting lasts from the beginning of July to the end of August. Almost 2000 hives stand on the Plateau at a time with a total yield of around 30,000 kg in a season, constituting about 0.03% of Turkey's total annual honey production (~100,000 tons/year).

Physicochemical Parameters

The average physicochemical parameters of honey samples collected at the beginning (CT1) and end of the harvesting times (CT2) were statistically different except for the moisture content (Tab. 1). They were also determined to be free from naphthalene, pesticides and antibiotics. All parameters complied with the Turkish Food Codex (2020) and Council Directive (2001). The compliance reveals that the samples carry the traits of safe and ordinary organic honey.

Colony Forming Units

The number of S. aureus colonies was 6.4×10³ cfu/g on agar gels before the incubation. After the incubation, circular zones formed around the wells on the agar gels for all phenol and honey samples, and not for negative control samples. The phenol samples underwent a ZD enlarging from 13.06 mm to 26.26 mm with increasing phenol concentration from 2% to 8%.

Zones of honey samples were in the form of two concentric circles, a wide inner zone and an enclosing thin halo zone. Honey samples exhibited an inner zone diameter ranging from 17.01 mm to 23.89 mm. The number of colonies was zero within the inner zones. On average, it was 15 cfu/g in the halo zones and 2.18×10¹⁰ cfu/g beyond the zones. The calibration curve (Fig. 2) exhibited a strong correlation with a coefficient of determination (R²) of 0.9957.

Statistics

Multidirectional statistical comparisons were performed for significant differences between groups. The difference between three HCs was insignificant in terms of ZD and TA values in all five hives at both CTs. The difference between five hives was insignificant at both CTs. The difference between the two CTs was also insignificant for ZD and TA values. Based on the inferential statistical analysis, all 60 values (two CTs × 30 samples) were gathered under one group.

Physicochemical Parameters

The honey mixtures exhibited statistically significant differences almost in terms of all physicochemical parameters at CT1 and CT2 (Tab. 1). The samples collected at CT1 consist of honey made between the period of mid-May and the beginning of July, and the samples collected at CT2 consist of honey made almost at the end of the nectar-flow period. The flora composition and weather are different between these periods on the Plateau. Every parameter of both honey mixtures complies with related regulations for honey (Turkish Food Codex, 2020; Council Directive, 2001).

Inhibition Zones and TA Values

Various agents are credited effective in varying degrees for the AMA of honey (Szweda, 2017). Among them, H₂O₂ is the dominant one for most honeys, forming through the catalyzes of glucose by glucose oxidase, and its concentration exhibits a strong correlation with TA (Guttentag et al., 2021). Two circular zones were observed for honey samples. The inner inhibition zone (first front) formed between the well and where MIC was reached. The agents had a sufficient microbiocidal concentration (SMC) in the inner zone to decrease the number of colonies from 6.4×10³ cfu/g before the incubation to null after the incubation. The halo zone formed between the first front and the second front (NIC). TA values for honey samples were determined from the inner zone diameter used as the ZD.

Statistical Assessment of TA Values

TA values of honey samples ranged between 14 and 27 with a range of 13, standard deviation of 3, mean of 21, median of 22, mode of 23–24, and Levene statistics of 0.380 (Fig. 4). Almost 95% of TA values were within the range of ±2 standard deviations of the mean (15≤21≤27), implying a normal distribution. The skewness of −0.525 is between −0.5 and −1.0, indicating that the distribution is moderately tilted to the right. The kurtosis of 2.417 (>+1) means the distribution is leptokurtic with thinner tails, indicating fewer outliers.

Physical Assessment of TA Values

The total AMA of honey is categorized as undetectable for TA<5, low for 5≤TA<10, therapeutically beneficial for TA≥10, and therapeutically high for TA≥20 (Carter et al., 2010). All honey samples from the Egricayır Plateau had a TA>10 and the minimum TA was 14. Most of the TA values (43 out of 60, 72%) were ≥20, which points to a high therapeutic potency. The distribution of TA values shows that the Egricayir Plateau is statistically and practically a homogeneous honey source in terms of TA for the season of 2023.

Comparison with Literature

TA of honey samples from the Egricayir Plateau was comparable with the literature. Green et al. (2022) determined the TA of thirty mānuka honey from Australia and New Zealand, of which 53% were therapeutic with an average TA of 19. Irish et al. (2011) worked on 477 Australian honey samples; more than half (57%) were therapeutic with a median TA of 16. Allen et al. (1991) examined 345 honey samples throughout New Zealand; 47% were therapeutic with an average TA of 19.

Honey samples from the Plateau are in the same category as high-potency honey (Green et al., 2023). They have a more homogeneous TA distribution and higher incidence of therapeutic AMA compared to the literature — 100% incidence of therapeutic AMA. The small forage territory should probably help with such a high incidence. For a consistent AMA and solid geographical indication, small territories seem more manageable than large ones at the expense of the tonnage.