Divergent polyphenolic modulation of glucose oxidase activity across honey types: Implications for medical-grade honey standardization

TL;DR

This study shows that polyphenols in honey affect the activity of the glucose oxidase enzyme, which is important for honey's antimicrobial properties, and suggests ways to standardize medical-grade honey.

Contribution

The study reveals divergent polyphenolic modulation of glucose oxidase activity across honey types and identifies biochemical mechanisms for standardization.

Findings

GOX activity varies significantly by honey type, with mixed and honeydew honeys showing higher activity than blossom honeys.

Polyphenolic content has opposite effects on GOX activity in blossom and honeydew honeys.

Low-molecular-weight phytochemicals act as non-protein cofactors that enhance GOX catalytic efficiency.

Abstract

Glucose oxidase (GOX)-mediated hydrogen peroxide (H2O2) generation constitutes a critical determinant of honey's antimicrobial efficacy, yet biochemical mechanisms governing botanical-origin-dependent variations remain incompletely elucidated. Our previous immunodetection analysis revealed no variation in GOX protein abundance among honeys, indicating post-secretion biochemical modulation rather than differential enzyme deposition. This study investigated relationships among GOX enzymatic activity, total polyphenolic content (TPPC), colour intensity, and antimicrobial properties across 99 unprocessed Slovak honey samples representing three botanical classifications: blossom (n = 36), mixed blossom-honeydew (n = 23), and honeydew (n = 40) honeys. GOX activity demonstrated marked botanical origin-dependent variation, with mixed and honeydew honeys exhibiting significantly elevated enzymatic activity compared to blossom honeys (P < 0.001). Critically, TPPC exerted divergent modulatory effects on GOX catalytic efficiency: positive correlation in blossom honeys (rs = 0.69, P < 0.001) versus inverse correlation in honeydew honeys (rs = −0.38, P < 0.05). Optimal enzymatic activity occurred at TPPC concentrations of 40-50 mg gallic acid equivalents/100 g, suggesting threshold-dependent biochemical regulation. High-temperature thermal processing (100 °C, 15 min) completely abolished GOX activity through irreversible structural disruption of both enzyme and thermolabile phytochemicals. Proteinase K digestion followed by reconstitution with exogenous fungal GOX demonstrated significant activity enhancement (37-92%) in honeydew samples compared to controls, confirming phytochemical-mediated catalytic augmentation. Dialysis experiments (MWCO 3500 Da) revealed that honeydew honeys maintained GOX protein levels while losing enzymatic activity, providing definitive evidence that low-molecular-weight phytochemicals function as non-protein cofactors modulating catalytic efficiency. These findings establish that honey polyphenolic compounds critically regulate GOX-mediated H2O2 production, with implications for developing evidence-based quality control parameters for medical-grade honey standardization.

Image 1

•GOX enzyme abundance is uniform; activity varies via phytochemical modulation.•Optimal enzymatic activity occurs at 40-50 mg GAE/100 g polyphenolic content.•Low-MW phytochemicals function as non-protein cofactors enhancing catalysis.•TPPC and GOX activity can serve as quality biomarkers for therapeutic honey.

GOX enzyme abundance is uniform; activity varies via phytochemical modulation.

Optimal enzymatic activity occurs at 40-50 mg GAE/100 g polyphenolic content.

Low-MW phytochemicals function as non-protein cofactors enhancing catalysis.

TPPC and GOX activity can serve as quality biomarkers for therapeutic honey.

Figures

Physicochemical and bioactive characteristics of honey samples classified by honey type. (A) Honey colour intensity expressed as Pfund values (mm). (B) Total polyphenolic content (TPPC) expressed as gallic acid equivalents (mg GAE/100 g). (C) Glucose oxidase (GOX) enzymatic activity (mU/ml) measured in 20% (w/v) honey solutions. (D) Minimum inhibitory concentration (MIC) against *Staphylococcus aureus* expressed as percentage (w/w %). Honey samples were classified into three groups based on electrical conductivity: blossom honey (n = 36), mixed blossom-honeydew honey (n = 23), and honeydew hon

Multivariate analysis and correlation patterns among honey characteristics. (A) Bubble plot showing the relationship between glucose oxidase (GOX) activity (mU/ml) and total polyphenolic content (TPPC, mg GAE/100 g). Bubble size represents electrical conductivity (EC, μS/cm) and colour gradient indicates Pfund values (mm), ranging from light (yellow, low Pfund) to dark (purple, high Pfund) honey. Dashed circle highlights blossom honey samples characterized by low TPPC and low GOX activity. (B) Correlation matrix heatmap displaying Spearman correlation coefficients (rs) between measured paramet

Effects of thermal treatment and proteolytic digestion on glucose oxidase activity in selected honey samples. (A) GOX activity following high-temperature thermal treatment (100 °C, 15 min) with or without reconstitution using exogenous fungal GOX (fGOX). (B) GOX activity following moderate thermal treatment (70 °C, 15 min) with or without exogenous fGOX addition. (C) GOX activity following proteinase K (ProtK) digestion, phenylmethylsulfonyl fluoride (PMSF) inactivation, and subsequent fGOX reconstitution. Selected honey samples include blossom honeys (No. 78, 82), mixed honeys (No. 86, 87), a

Protein profiles and glucose oxidase immunodetection in honey samples following proteinase K treatment. (A) SDS-PAGE analysis showing protein profiles of selected honey samples (blossom: No. 78, 82; mixed: No. 86, 87; honeydew: No. 96, 98) with (+) or without (−) proteinase K (PK) digestion. M, molecular weight marker. (B) Western blot analysis of the same samples using polyclonal anti-GOX antibody. Proteinase K treatment resulted in complete protein degradation in most samples (78, 82, 86, 87), while honeydew samples (96, 98) retained residual undigested protein bands following proteolytic di

Effects of dialysis on glucose oxidase activity and protein content in honey samples. (A) GOX enzymatic activity (mU/ml) measured in control honey (gray bars, untreated 50% w/w solutions), cold-treated honey (blue bars, incubated overnight at 4 °C), and dialysed honey (red bars, dialyzed using 3500 Da MWCO cassettes overnight at 4 °C). (B) Semi-quantitative analysis of GOX protein content determined by densitometric analysis of immunoreactive bands from Western blots, expressed as band intensity (intensity × mm^2^). Selected honey samples: blossom (No. 78, 82), mixed (No. 86, 87), and honeydew

Full text

Introduction

1

Honey is a natural product highly valued for its nutritional, medicinal and sensory properties. Traditionally, honey quality is assessed according to Codex Alimentarius standards (Codex Alimentarius, 2001) and/or European Union Honey Directive (EU Directive, 2002), utilizing a defined set of qualitative parameters, including moisture content, sugar composition, acidity, diastase activity and hydroxymethylfurfural (HMF) content. However, escalating consumer demand for authentic honey with demonstrated health benefits (Ioniț;mă-Mîndrican et al., 2022; Zanchini et al., 2022), coupled with intensifying concerns over food fraud, has created an urgent need for more sophisticated, reliable, and specific quality markers in honey research. This necessitates a critical reconsideration of current quality parameters (Majtan, 2024). Moreover, the development of rapid, cost-effective and bench-top analytical methods that accurately reflect the biological properties of honey need to be developed (Álvarez-Suárez and Majtan, 2026).

Honey exhibits well-described antibacterial activity mediated through multiple biochemical and biophysical mechanisms (Majtan et al., 2021). Most antibacterial compounds in honey to date target bacterial cells non-specifically, including the principal bactericidal compounds, hydrogen peroxide (H_2_O_2_) and methylglyoxal (MGO). The concentrations of these compounds correlate with honey's antibacterial potency, with minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) ranging from 10 to 1000 μg/ml. Nevertheless, conflicting conclusions have emerged from different studies regarding the correlation between antimicrobial activity and H_2_O_2_ content. In an extensive investigation analysing 233 samples (Farkasovska et al., 2019), acacia, rapeseed, sunflower and multi-floral honeys demonstrated moderate correlations between antibacterial activity and H_2_O_2_ content. Conversely, linden honey exhibited the strongest antibacterial effect while showing weak or no correlation between antibacterial activity and H_2_O_2_ levels (Farkasovska et al., 2019). Similarly, honeydew honey demonstrated a distinct H_2_O_2_-generating mechanism that may not depend exclusively on glucose oxidase (GOX) enzymatic activity (Bucekova et al., 2018; Bucekova et al., 2014). Honeydew honeys contain elevated levels of phenolic acids and flavonoids with potent antioxidant and pro-oxidant properties. Recent research (Sanhueza and Fuentes, 2025) has demonstrated that flavonoids, particularly flavonols, contribute to H_2_O_2_ generation to a lesser extent than GOX activity. Consequently, the bee-derived GOX enzyme, as a ubiquitous component of all honey types, remains the predominant producer of H_2_O_2_ in diluted honey solutions (White et al., 1963).

H_2_O_2_ concentration represents a sensitive parameter influenced by multiple factors. A critical question concerns the optimal incubation time required for samples to achieve relevant H_2_O_2_ production. A recent investigation of honeys from New Caledonia revealed divergent kinetics of H_2_O_2_ generation after 24 h in 40% diluted solutions (Bucekova et al., 2023a). When honeys exhibiting the highest and lowest H_2_O_2_ concentrations were selected, and production kinetics were monitored at MIC concentrations (6% diluted honey), a pronounced shift in the enzymatic kinetic profile of H_2_O_2_ production among samples was observed.

The final H_2_O_2_ concentration may be significantly affected by the presence of plant-derived catalase (CAT) in honey. Simultaneous determination of GOX and CAT activities in honeys has been performed in only a limited number of studies (Alshareef et al., 2022; Osés et al., 2024), yielding contradictory outcomes. Brudzynski (2020) (Brudzynski, 2020) comprehensively reviewed the relationship between H_2_O_2_ variation and multiple factors, including variable quantities of GOX produced by bees (related to bee age, caste, and pollen nutrition), glucose concentration, water activity, and osmolarity. Elevated glucose concentration and osmolarity combined with low water activity reduce molecular mobility and thereby inhibit the GOX reaction. Additional factors affecting H_2_O_2_ levels include H_2_O_2_ accumulation dynamics, photosensitivity and thermosensitivity of H_2_O_2_, CAT activity, decomposition by metal-containing enzymes and ascorbic acid, products derived from polyphenol autoxidation, presence of GOX in some flowers, and H_2_O_2_ production by microorganisms present in honey. Furthermore, divergent methodological approaches (fluorometric vs. spectrophotometric) and experimental conditions for determining GOX activity and H_2_O_2_ concentration in diluted honeys complicate direct inter-study comparison.

Therefore, this study aimed to: (i) assess GOX enzymatic activity across different honey types using a robust spectrophotometric method; (ii) quantify colour intensity and total polyphenolic content in all samples (n = 99) and perform correlation analysis to determine relationships between variables; (iii) evaluate antimicrobial activity against Staphylococcus aureus; and (iv) investigate the role of phytochemicals in modulating GOX activity using thermal treatment and dialysis separation techniques.

Materials and methods

2

Honey samples

2.1

In total, 99 unprocessed honey samples collected throughout 2022 were supplied directly by beekeepers from Slovakia. Upon arrival, samples were immediately stored in plastic containers at 4 °C in the dark. The samples were classified according to their electrical conductivity values (Bogdanov, 2002; Codex Alimentarius, 2001) and categorised into three major groups: blossom honey <500 μS/cm (n = 36), mixed blossom-honeydew honey 500-800 μS/cm (n = 23), and pure honeydew honey >800 μS/cm (n = 40).

As a negative control artificial honey (AH) was prepared by dissolving 39 g d-fructose, 31 g, d-glucose, 8 g maltose, 3 g sucrose and 19 g distilled water, as described elsewhere (Majtan and Majtan, 2010).

Bacteria

2.2

The antibacterial activity of honey samples was assessed against the model pathogenic bacteria Staphylococcus aureus CCM4223, obtained from the Department of Medical Microbiology, Slovak Medical University (Bratislava, Slovakia).

Determination of antibacterial activity

2.3

The antibacterial efficacy of the honey samples was evaluated with a minimum inhibitory concentration (MIC) assay as described by Bucekova et al. (2023b). Briefly, overnight bacterial culture was suspended in phosphate-buffered saline (PBS), pH 7.2, and the turbidity of the suspension was adjusted to 10^8^ colony-forming unit (CFU)/ml and diluted with Mueller-Hinton broth (MHB) medium (pH 7.3 ± 0.1) to a final concentration of 10^6^ CFU/ml. Then, 10- μl aliquots of suspension were inoculated into each well of sterile 96-well polystyrene plates (Sarstedt, Germany). The final volume in each well was 100 μl, consisting of 90 μl of sterile medium or diluted honey and 10 μl of bacterial suspension. After 18 h of incubation at 37 °C, bacterial growth inhibition was determined by visual inspection. The MIC was defined as the lowest concentration of honey inhibiting bacterial growth. All tests were performed in triplicate and repeated three times. Serial dilutions of each honey sample were prepared from a 50% (w/v) honey solution, resulting in final concentrations of 45, 36, 18, 9 and 4.5%.

Assessment of honey colour according to pfund method

2.4

For visual assessment, 50% (w/w) honey solutions were prepared and heated at 50 °C for 10 min. Subsequently, absorbances were measured using UV/V spectrophotometer (Thermo Electronic) and a wave scan for 635 nm in disposable plastic cuvettes with 10 nm optical pathlength. Sterile water was used as a blank. Honey colour was than assessed using Pfund value calculated according following equation (Ferreira et al., 2009):

$$[eqn]$$

Assessment of total polyphenolic content

2.5

Total phenolic content was determined with a Folin Ciocalteu Phenolic Content Quantification Assay Kit (BioQuoChem, Spain) in a 20% (w/v) honey solution in a 96-well microplate according to the manufacturer's instructions. Gallic acid equivalents (GAE) were used as the reference standard and results were expressed as GAE equivalents (mg/ml). Absorbance was measured at 700 nm at room temperature.

Determination of GOX activity

2.6

Bee-derived GOX activity was determined using a Megazyme GOX assay kit (Megazyme International Ireland Ltd., Bray, Ireland), which is based on the oxidative catalysis of β-D-glucose to D-glucono-δ-lactone, with the concurrent release of H_2_O_2_. The resultant H_2_O_2_ reacts with p-hydroxybenzoic acid and 4-aminoantipyrine in the presence of peroxidase to form a quinoneimine dye complex, which has a strong absorbance at 510 nm. Enzyme activity was determined in a 96-well microplate according to the manufacturer's instructions. Activity was assessed in freshly prepared 20% (w/v) honey solutions, which were centrifuged (10000 rpm, 5 min, RT) to remove insoluble particles. The microplates were assayed immediately and after incubation in the dark for 20 min.

Heat, proteolytic and dialysis treatment of honey samples

2.7

To characterise the potential effect of phytochemicals on GOX enzymatic activity, honey samples with different botanical origins with EC varied from 163 to 1363 μS/cm and GOX activity 2.83 - 30.75 mU/ml (No. 78, 82, 86, 87, 96, and 98) were subjected to different treatment procedures to characterise the effect of phytochemical content on GOX enzymatic activity:

• a)Heat treatment. Honey samples were heated either at 100 °C or 70 °C for 15 min and subsequently cooled to room temperature. Commercially available purified fungal GOX enzyme (Magazine International Ireland Ltd., 0.58 U/ml) was added to heat-treated honey samples, and GOX enzymatic activity was measured in 20% honey solutions. Fungal GOX enzyme in artificial honey was used as a positive control, with a final enzymatic activity of 15 mU/ml in a 20% artificial honey solution.
• b)Proteolytic treatment. Diluted honey samples (40% w/w in PBS, pH 7.2) were treated with proteinase K (30 U/mg; Promega) at a final concentration of 50 μg/mL at 37 °C for 30 min. Subsequently, proteolytic activity was terminated by incubating the mixture with 1 mM phenylmethylsulfonyl fluoride (PMSF) at room temperature for 30 min. Finally, the fungal GOX enzyme was added to each conditioned honey sample. Fungal GOX enzyme in artificial honey served as a positive control, with a final enzymatic activity of 29 mU/ml in a 20% artificial honey solution.
• c)Dialysis treatment. Low molecular weight honey compounds, including polyphenols, were removed from 15 ml of 50% (w/w) honey solutions by dialysis using Pur-A-Lyzer MEGA 3500 dialysis cassettes with a molecular weight cut-off of 3500 Da (Sigma-Aldrich, St. Luis, MO, USA) against 4 L of distilled water overnight at 4 °C. Control honey samples consisted of 15 ml of 50% honey solutions incubated overnight at 4 °C without dialysis. GOX enzymatic activity and GOX content evaluated by immunoblotting were determined in all dialysed and non-dialysed honey samples adjusted to equal volumes.

Proteins profile of honey samples and GOX enzyme detection by immunoblotting

2.8

For protein determination, 15 μL aliquots of diluted honey samples (50 % w/w in distilled water) were loaded on 12 % SDS-PAGE gels and separated using a Mini-Protean II electrophoresis cell (Bio-Rad, Hercules, CA, USA). Protein content was assessed after gel staining with Coomassie Brilliant Blue R-250 (Sigma-Aldrich, Darmstadt, Germany).

For immunodetection of GOX enzyme, proteins separated on SDS-PAGE gels were transferred onto 0.22 μm nitrocellulose Advantec membrane (Sigma-Aldrich) in transfer buffer (48 mM Tris, 39 mM glycine, and 20% methanol) using a wet blotting procedure. Membranes were blocked for 1 h in Tris-buffered saline-Tween (TBST) buffer (50 mM Tris–HCl, pH 7.5, 200 mM NaCl and 0.05% Tween 20) containing 5% non-fat dried milk and incubated overnight with rabbit polyclonal antibody against honeybee GOX (1:2000 in TBST (Bucekova et al., 2018). After washing with TBST, membranes were incubated for 2 h in blocking buffer containing goat anti-rabbit horseradish peroxidase-linked antibodies (1:2500 in TBST; Promega). Immunoreactive bands were detected using SigmaFast 3,3-diaminobenzidine tablets (Sigma-Aldrich) dissolved in solution. Membranes were scanned, and immunoreactive bands were analysed and semi-quantified by densitometry using ImageJ software (NIH, version 1.52a, Bethesda, MD, USA).

Statistical analysis and figure preparation

2.9

Whether data were normally distributed was determined using the Shapiro-Wilk normality test, followed by ANOVA and post-hoc Tukey's test to analyse the significance between groups. According to sampled data distribution, Pearson or nonparametric Spearman correlation was computed and visualized using a correlation matrix with multiple variables and simple correlation using two variables.

Multivariate analysis was used to assess differences between variables and Principal Component Analysis was illustrated on bubble plots created directly from raw data. P values < 0.05 were considered significant. Data were analysed using Prism 10 (GraphPad Software Inc., La Jolla, CA, USA)

Results

3

Physico-chemical and biochemical characteristics of honey samples

3.1

Electrical conductivity, honey color, GOX enzymatic activity, and TPPC were determined in all honey samples, which were classified as blossom (BH; n = 36), blossom-honeydew (MH; n = 23), and honeydew (HH; n = 40) honey samples.

The range of color in the sample set extended from the lightest water white (−14.56 mm Pfund) in sample no. 77 to the darkest (122.9 mm Pfund) in sample no. 68 (Table 1). Water white color was found in 11.11% of samples, followed by extra white in 5.05% of samples, and white in 18.20%. Extra light amber represented 19.20%, and the largest portion of honey samples exhibited light amber color in 32.20% of samples. Amber and dark amber were found in 12.10% and 2.0% of samples, respectively. As expected, the lighter-colored honeys were in BH group, where Pfund values varied from −14.56 mm to 52.22 mm with a mean value of 17.94 ± 18.19 mm. The MH and HH groups were significantly darker (P < 0.001) than the BH group (Fig. 1A). Interestingly, there was no significant difference between the MH and HH groups in color. The recorded maximal and minimal values of both groups were similar. The maximal and minimal color value were 121.0 and 22.95 mm Pfund for the MH group and 122.9 and 14.78 mm Pfund for the HH group, respectively. The mean values were 57.52 ± 22.41 mm Pfund for the MH group and 68.72 ± 22.33 mm Pfund for the HH group.Table 1. Biochemical, physicochemical, and antibacterial characteristics of honey samples (n = 99).Table 1. Sample no.EC (μS/cm)GOX activity (mU/mL)TPPC (mg GAE/100 g)MIC (%)Pfund (mm)1728.00 ± 1.008.65 ± 2.3232.49 ± 2.4345.00 ± 0.0097.232777.00 ± 1.0019.06 ± 3.2444.35 ± 4.114.50 ± 0.0071.973660.00 ± 0.0027.55 ± 3.6452.05 ± 7.254.50 ± 0.0075.324639.00 ± 0.0017.11 ± 1.8138.13 ± 1.354.50 ± 0.0061.585925.00 ± 1.0022.84 ± 2.3147.32 ± 3.279.00 ± 0.0039.666792.00 ± 0.0016.21 ± 2.2052.67 ± 6.574.50 ± 0.0088.327669.00 ± 0.0014.47 ± 1.7539.06 ± 1.474.50 ± 0.0060.0981238.00 ± 2.0019.92 ± 4.4052.18 ± 4.8733.00 ± 4.2474.959240.40 ± 0.004.28 ± 0.8830.09 ± 3.7338.00 ± 2.8352.6610157.00 ± 0.000.72 ± 0.5123.48 ± 2.609.00 ± 0.0039.2911174.00 ± 0.003.76 ± 2.3619.39 ± 3.069.00 ± 0.0020.7212517.00 ± 1.0016.64 ± 3.2336.34 ± 2.054.50 ± 0.0028.1513470.00 ± 0.006.44 ± 1.8828.64 ± 1.694.50 ± 0.0045.2314743.00 ± 1.0023.23 ± 4.9739.70 ± 3.674.50 ± 0.0063.4315720.00 ± 0.0012.73 ± 3.1944.23 ± 1.704.50 ± 0.0052.6616784.00 ± 0.0016.94 ± 4.3143.84 ± 9.294.50 ± 0.0042.2617998.00 ± 2.0021.73 ± 3.0846.41 ± 4.989.00 ± 0.0092.40181060.00 ± 2.0016.47 ± 2.0851.69 ± 8.7336.00 ± 0.00102.8019437.00 ± 0.0015.65 ± 3.3630.05 ± 4.424.50 ± 0.0037.06201090.00 ± 1.0010.16 ± 1.1160.47 ± 3.3128.00 ± 11.3195.0021183.80 ± 0.000.00 ± 0.0020.32 ± 2.2840.00 ± 0.000.3022196.00 ± 0.005.23 ± 1.3728.38 ± 5.2735.50 ± 0.7120.7223650.00 ± 1.0016.07 ± 3.3038.13 ± 4.024.50 ± 0.0037.0624674.00 ± 0.0013.85 ± 1.8638.10 ± 2.199.00 ± 0.0056.3825928.00 ± 2.0022.03 ± 3.7050.00 ± 3.914.50 ± 0.0069.7526890.00 ± 1.0017.42 ± 3.7846.74 ± 9.344.50 ± 0.0056.3827422.00 ± 0.0010.39 ± 1.7130.83 ± 6.919.00 ± 0.0039.2928304.00 ± 0.0010.14 ± 1.7130.88 ± 3.914.50 ± 0.0010.6929471.00 ± 0.005.36 ± 1.3330.83 ± 3.869.00 ± 0.0022.2130480.00 ± 0.0012.26 ± 1.6731.66 ± 6.449.00 ± 0.0028.8931343.00 ± 0.007.36 ± 2.8727.88 ± 2.224.50 ± 0.0045.98321111.00 ± 3.0031.91 ± 3.2049.11 ± 5.844.50 ± 0.0073.8333518.00 ± 1.0020.82 ± 4.8936.72 ± 2.859.00 ± 0.0054.1534196.80 ± 0.006.94 ± 2.8724.99 ± 1.669.00 ± 0.0021.0935705.00 ± 0.0021.01 ± 2.7532.48 ± 3.2235.50 ± 0.7145.9836447.00 ± 0.0021.82 ± 3.8037.42 ± 5.769.00 ± 0.0043.7537449.00 ± 0.002.31 ± 1.9722.93 ± 2.5118.00 ± 0.0014.0438719.00 ± 0.0014.88 ± 1.1337.31 ± 6.8435.50 ± 0.0042.2639525.00 ± 0.009.76 ± 2.8733.27 ± 3.329.00 ± 0.0022.9540323.00 ± 0.0013.72 ± 4.0925.21 ± 6.559.00 ± 0.0031.12411155.00 ± 2.0021.81 ± 1.7180.71 ± 5.854.50 ± 0.00102.8042988.00 ± 2.0010.55 ± 1.2763.66 ± 10.334.50 ± 0.0074.57431198.00 ± 4.0022.27 ± 1.5955.09 ± 7.734.50 ± 0.0056.00441232.00 ± 1.0017.03 ± 1.1370.96 ± 4.064.50 ± 0.0072.3545997.00 ± 0.0022.33 ± 1.7140.10 ± 4.8130.50 ± 7.7814.7846658.00 ± 0.007.47 ± 1.4043.42 ± 5.9236.00 ± 0.0061.9547545.00 ± 0.007.40 ± 3.1340.68 ± 5.954.50 ± 0.0056.38481027.00 ± 1.0013.76 ± 2.0865.19 ± 4.264.50 ± 0.0075.3249848.00 ± 0.0011.07 ± 1.9646.60 ± 3.504.50 ± 0.0054.89501097.00 ± 1.0015.49 ± 1.5352.49 ± 7.534.50 ± 0.0088.3251465.00 ± 0.0017.62 ± 2.4147.12 ± 6.2430.50 ± 7.7822.58521318.00 ± 2.0016.33 ± 2.1242.21 ± 8.674.50 ± 0.0024.44531215.00 ± 1.0013.05 ± 1.3974.09 ± 10.964.50 ± 0.0096.49541116.00 ± 1.0019.54 ± 1.1647.66 ± 5.3736.00 ± 0.045.9855595.00 ± 0.0017.03 ± 3.9939.46 ± 2.724.50 ± 0.0034.0956333.00 ± 0.009.71 ± 2.7932.65 ± 2.284.50 ± 0.0024.06571054.00 ± 1.0017.44 ± 1.7250.31 ± 4.754.50 ± 0.0050.43581265.00 ± 2.0015.32 ± 1.0165.76 ± 4.954.50 ± 0.0064.55591458.00 ± 4.008.44 ± 1.6063.01 ± 4.584.50 ± 0.0048.21601042.00 ± 3.0015.24 ± 2.2259.79 ± 3.014.50 ± 0.0084.2361194.20 ± 0.005.38 ± 2.0819.57 ± 0.849.00 ± 0.00−1.9362179.50 ± 0.004.78 ± 2.5122.63 ± 2.469.00 ± 0.0013.6763334.00 ± 0.0014.95 ± 5.8834.26 ± 5.814.50 ± 0.0030.0164153.70 ± 0.005.95 ± 2.7518.69 ± 1.589.00 ± 0.00−6.39651275.00 ± 1.0016.47 ± 2.4558.33 ± 4.744.50 ± 0.0049.32661322.00 ± 4.0017.35 ± 1.2444.50 ± 6.314.50 ± 0.0060.0967605.00 ± 0.0016.45 ± 3.8856.48 ± 15.624.50 ± 0.0056.0068908.00 ± 0.0014.03 ± 4.0355.56 ± 5.544.50 ± 0.00122.85691190.00 ± 2.0012.75 ± 2.9348.93 ± 2.404.50 ± 0.0042.6370399.00 ± 0.008.50 ± 3.6330.96 ± 1.859.00 ± 0.0027.41711085.00 ± 1.0021.39 ± 1.6050.13 ± 3.874.50 ± 0.0074.57721300.00 ± 1.0016.89 ± 2.5151.86 ± 0.764.50 ± 0.0067.15731292.00 ± 2.0016.22 ± 2.8465.01 ± 1.854.50 ± 0.0066.7774282.00 ± 0.008.28 ± 1.8533.35 ± 4.049.00 ± 0.00−3.05751092.00 ± 3.0015.73 ± 1.6352.10 ± 3.624.50 ± 0.0094.2676243.40 ± 0.0010.85 ± 2.6225.07 ± 1.774.50 ± 0.0027.7877128.70 ± 0.005.23 ± 1.5915.61 ± 1.019.00 ± 0.00−14.5678198.50 ± 0.002.83 ± 1.6725.61 ± 1.689.00 ± 0.006.2479143.80 ± 0.000.94 ± 0.7615.37 ± 0.8417.00 ± 1.41−13.0780164.70 ± 0.004.03 ± 1.4419.89 ± 1.359.00 ± 0.00−10.1081149.90 ± 0.003.52 ± 1.2320.91 ± 1.099.00 ± 0.00−9.7382163.10 ± 0.006.07 ± 2.3117.90 ± 4.409.00 ± 0.00−4.1683156.20 ± 0.003.72 ± 3.6617.84 ± 2.5921.50 ± 4.9518.8784170.00 ± 0.003.22 ± 1.6518.88 ± 2.5016.00 ± 2.8320.7285195.00 ± 0.004.70 ± 1.7622.66 ± 3.549.00 ± 0.0021.0986808.00 ± 0.0012.70 ± 7.2236.50 ± 0.524.50 ± 0.0048.2187865.00 ± 1.0027.25 ± 4.9847.35 ± 2.934.50 ± 0.0066.4088207.50 ± 0.004.19 ± 1.6724.12 ± 3.369.00 ± 0.0016.6489814.00 ± 0.0013.89 ± 2.6048.09 ± 4.484.50 ± 0.0095.0090424.00 ± 0.007.33 ± 2.9721.87 ± 2.419.00 ± 0.006.6191935.00 ± 2.0018.51 ± 1.1741.07 ± 3.714.50 ± 0.0064.1892691.00 ± 0.0015.32 ± 3.9131.99 ± 3.104.50 ± 0.0046.3593728.00 ± 0.0012.79 ± 3.2636.07 ± 4.744.50 ± 0.0047.46941175.00 ± 2.009.69 ± 1.7646.87 ± 4.314.50 ± 0.0073.83951362.00 ± 3.0020.02 ± 1.6456.87 ± 4.484.50 ± 0.0087.94961071.00 ± 1.0023.59 ± 2.5653.45 ± 7.594.50 ± 0.0071.9797778.00 ± 0.0011.61 ± 1.9355.46 ± 0.604.50 ± 0.00121.00981363.00 ± 1.0030.75 ± 2.2534.73 ± 0.099.00 ± 0.0051.55991557.00 ± 3.0010.97 ± 1.9674.67 ± 9.664.50 ± 0.0054.15Data represent mean ± standard deviation.Fig. 1. Physicochemical and bioactive characteristics of honey samples classified by honey type. (A) Honey colour intensity expressed as Pfund values (mm). (B) Total polyphenolic content (TPPC) expressed as gallic acid equivalents (mg GAE/100 g). (C) Glucose oxidase (GOX) enzymatic activity (mU/ml) measured in 20% (w/v) honey solutions. (D) Minimum inhibitory concentration (MIC) against Staphylococcus aureus expressed as percentage (w/w %). Honey samples were classified into three groups based on electrical conductivity: blossom honey (n = 36), mixed blossom-honeydew honey (n = 23), and honeydew honey (n = 40). Each data point represents an individual honey sample. Red horizontal lines indicate mean values. Statistical significance was determined using one-way ANOVA followed by Tukey's post-hoc test. ∗∗∗P < 0.001; ns, not significant.Fig. 1

The TPPC content in the samples varied from 15.37 to 80.71 mg GAE/100 g, with an average value of 40.58 ± 14.91 mg GAE/100 g (Table 1). TPPC reflected the compositional variability among honey groups. We observed significant differences among all three groups (P < 0.001) (Fig. 1B). In the BH group, TPPC varied from 15.37 to 47.12 mg GAE/100 g with a mean value of 25.78 ± 6.80 mg GAE/100 g. In the MH group, the TPPC content varied from 31.99 to 56.48 mg GAE/100 g with a mean value of 40.98 ± 7.197 mg GAE/100 g. The HH group consisted of samples with the highest concentrations of TPPC, ranging from 34.73 to 80.71 mg GAE/100 g with a mean value of 53.69 ± 10.44 mg GAE/100 g.

GOX enzymatic activity varied from the lowest value of 0 mU/ml (sample no. 21) to the highest value of 31.91 mU/ml (sample no. 32), with a mean value of 13.21 ± 7.02 mU/ml (Table 1). One-third (33.33%) of the samples had GOX activity lower than 10 mU/ml, while 49.5% of the samples exhibited GOX activity in the range of 10-20.00 mU/ml. The group with the highest GOX activity (20.00 to 31.9 mU/ml) was represented by 17.2% of the total samples. The mean value of GOX activity in the BH group was 7.02 ± 5.07 mU/ml. Interestingly, in the MH and HH group the lowest recorded GOX activities were higher than the mean value in BH group, representing 7.40 and 8.437 mU/ml, respectively (Fig. 1C). Therefore, both MH and HH groups showed significantly higher GOX activity (P < 0.001) than the BH group (Fig. 1C)

Antibacterial activity of honey samples

3.2

MIC values of 99 honey samples against the Gram-positive bacterium S. aureus are shown in Table 1. MIC values for the honey samples ranged from 4.5 to 45% with a mean value of 10.33 ± 10.37%. More than half of the tested samples had an MIC value of 4.5%. Overall, 82.8% of all tested samples had MIC values below 10%. One sample had an MIC of 45%, indicating antibacterial activity attributable solely to sugar compounds. The lowest measured MIC was 4.5% in all honey groups. The highest MIC was 40.0% in the BH group, with a mean of 12.14 ± 9.4% (Fig. 1D). In the MH group, the highest MIC value was 45.0% with a mean of 10.91 ± 12.91% and in the HH group, the maximum MIC was 36% with a mean value of 8.36 ± 9.46%. There was no significant difference between groups (Fig. 1D).

Mutual relations and correlations between GOX, TPPC, pfund and MIC in honey groups

3.3

Fig. 2A shows the multivariable analysis of all measured values in all samples analysed. There is an apparent cluster containing samples with the lowest TPPC, GOX activity, light watery colours, and the lowest EC values, which belong to the BH group. There rest of the samples are evenly distributed along axes with GOX activity values with rising TPPC and EC values.Fig. 2. Multivariate analysis and correlation patterns among honey characteristics. (A) Bubble plot showing the relationship between glucose oxidase (GOX) activity (mU/ml) and total polyphenolic content (TPPC, mg GAE/100 g). Bubble size represents electrical conductivity (EC, μS/cm) and colour gradient indicates Pfund values (mm), ranging from light (yellow, low Pfund) to dark (purple, high Pfund) honey. Dashed circle highlights blossom honey samples characterized by low TPPC and low GOX activity. (B) Correlation matrix heatmap displaying Spearman correlation coefficients (rs) between measured parameters: GOX activity, electrical conductivity (EC), total polyphenolic content (TPPC), colour intensity (Pfund), and minimum inhibitory concentration (MIC). Colour scale represents correlation strength from −1.0 (purple, negative correlation) to +1.0 (yellow, positive correlation). All honey samples (n = 99) were included in the analysis.Fig. 2

Correlation of variables was calculated using Spearman correlation and showed correlation matrix showing Spearman r values in Fig. 2B. The highest correlation was observed between TPPC and EC (r_s_ = 0.87), TPPC and colour (Pfund) (r_s_ = 0.84), followed by colour correlated with EC (r_s_ = 0.74) and GOX activity with EC (r_s_ = 0.67). Low negative correlation was observed between antibacterial activity (MIC) and all measured parameters.

Strong negative correlation (r_s_ = −0.53; P < 0.001) was observed between antibacterial activity and GOX activity only in the BH group (Fig. S1). There were no correlations observed between these two parameters in MH and HH. The BH group showed significant correlations of GOX activity with TPPC (r_s_ = 0.69; P < 0.001), antibacterial MIC value (r_s_ = −0.53; P < 0.001) and Pfund value (r_s_ = 0.49; P < 0.01) (Fig. S1). In the HH group, a significant correlation of GOX activity with TPPC (r_s_ = −0.38; P < 0.05) was observed. Interestingly, there was no significant correlation observed in the MH group of GOX activity with any of the measured parameters.

Contribution of phytochemicals to GOX enzymatic activity in honey samples

3.4

To characterise the potential effect of phytochemicals on GOX enzymatic activity, honey samples with different botanical origins (No. 78, 82, 86, 87, 96, and 98) were employed (Fig. 3).Fig. 3. Effects of thermal treatment and proteolytic digestion on glucose oxidase activity in selected honey samples. (A) GOX activity following high-temperature thermal treatment (100 °C, 15 min) with or without reconstitution using exogenous fungal GOX (fGOX). (B) GOX activity following moderate thermal treatment (70 °C, 15 min) with or without exogenous fGOX addition. (C) GOX activity following proteinase K (ProtK) digestion, phenylmethylsulfonyl fluoride (PMSF) inactivation, and subsequent fGOX reconstitution. Selected honey samples include blossom honeys (No. 78, 82), mixed honeys (No. 86, 87), and honeydew honeys (No. 96, 98). Artificial honey (AH) served as the negative control. Red dashed lines indicate reference GOX activity level (fGOX in artificial honey). Data represent mean ± standard deviation from three independent experiments. Statistical significance compared to fGOX control was determined using Student's t-test. ∗∗P < 0.01; ∗∗∗P < 0.001.Fig. 3

As anticipated, thermal treatment (100 °C, 15 min) resulted in complete abolishment of GOX activity across all honey samples evaluated (Fig. 3A). When exogenous fungal GOX enzyme, at equivalent concentrations, was reconstituted into thermally treated samples, GOX activity was only partially restored but remained significantly attenuated compared to the positive control (artificial honey fortified with fungal GOX), indicating that certain thermolabile phytochemicals or thermally induced formation of inhibitory compounds, including highly reactive quinones produced by the oxidation of flavonols may modulate GOX enzymatic activity in diluted honey matrices.

Moderate thermal treatment (70 °C, 15 min) demonstrated differential effects on GOX activity in honey samples, contingent upon honey types and the initial GOX activity of honey samples prior to thermal processing (Fig. 3B). Thermal treatment significantly diminished (P < 0.001; P < 0.01) endogenous GOX activity in all honey samples, with the exception of sample No. 87. Supplementation of thermally treated honey samples with exogenous fungal GOX enhanced enzymatic activity.

As demonstrated above, thermal processing at high temperature can exert deleterious effects on GOX activity as well as on thermolabile phytochemicals possessing GOX activity-enhancing properties. Therefore, we elucidated the role of phytochemicals in augmented GOX activity in honey samples by eliminating honey total proteinaceous content, including GOX enzyme, utilizing proteinase K digestion. Incubation of honey solutions with proteinase K resulted in complete proteolytic degradation of proteinaceous content, except for two samples No. 86 and 87, where residual undigested protein bands were visualized on SDS-PAGE gels (Fig. 4A). However, GOX enzyme underwent complete degradation with proteinase K in all honey samples (Fig. 4B). Following proteinase K inactivation by PMSF and subsequent reconstitution of honey samples and artificial honey with exogenous fungal GOX enzyme, a significant (P < 0.001) enhancement in GOX enzymatic activity was documented in four honey samples No. 86, 87, 96 and 98 by 36.7, 77.6, 46.7 and 92.2%, respectively, in comparison to artificial honey fortified with fungal GOX enzyme (Fig. 3C). Conversely, GOX enzymatic activity remained unaltered in sample No. 78 and 82 and was equivalent to the enzymatic activity of fungal GOX enzyme in artificial honey. Notably, both honey samples exhibited the diminished and lowest Pfund and TPPC values, respectively (Table 1).Fig. 4. Protein profiles and glucose oxidase immunodetection in honey samples following proteinase K treatment. (A) SDS-PAGE analysis showing protein profiles of selected honey samples (blossom: No. 78, 82; mixed: No. 86, 87; honeydew: No. 96, 98) with (+) or without (−) proteinase K (PK) digestion. M, molecular weight marker. (B) Western blot analysis of the same samples using polyclonal anti-GOX antibody. Proteinase K treatment resulted in complete protein degradation in most samples (78, 82, 86, 87), while honeydew samples (96, 98) retained residual undigested protein bands following proteolytic digestion.Fig. 4

Considering the low MW of honey phytochemicals ranging from 200 to 500 Da and the substantially higher MW (160 kDa) of the GOX enzyme, dialysis of honey samples was performed using dialysis cassettes with a MW cut-off of 3500 Da. GOX activity of dialysed honey samples was significantly (P < 0.001) attenuated across all samples (Fig. 5A). Incubation of honey sample solutions at 4 °C without dialysis did not alter GOX activity in any samples. The observed attenuation of GOX activity is predominantly attributed to GOX aggregation during dialysis, as demonstrated in Fig. 5B, where a substantial reduction in GOX abundance was documented. However, the GOX enzyme content in dialysed honey samples No. 96 and 98 (honeydew honeys) remained unaltered compared to unprocessed honey samples (Fig. 5B), substantiating the significant role of phytochemicals in the enhancement of GOX activity in honey matrices.Fig. 5. Effects of dialysis on glucose oxidase activity and protein content in honey samples. (A) GOX enzymatic activity (mU/ml) measured in control honey (gray bars, untreated 50% w/w solutions), cold-treated honey (blue bars, incubated overnight at 4 °C), and dialysed honey (red bars, dialyzed using 3500 Da MWCO cassettes overnight at 4 °C). (B) Semi-quantitative analysis of GOX protein content determined by densitometric analysis of immunoreactive bands from Western blots, expressed as band intensity (intensity × mm^2^). Selected honey samples: blossom (No. 78, 82), mixed (No. 86, 87), and honeydew (No. 96, 98). Data represent mean ± standard deviation from three independent experiments. Statistical significance compared to control honey was determined using one-way ANOVA followed by Tukey's post-hoc test. ∗P < 0.05; ∗∗∗P < 0.001.Fig. 5

Discussion

4

The GOX enzyme represents the principal enzymatic catalyst mediating H_2_O_2_ generation in aqueous honey solutions. While the concentration of accumulated H_2_O_2_ remains relatively low, it confers potent antimicrobial efficacy, exhibiting both bacteriostatic and bactericidal properties against pathogenic bacterial species. Our recent investigations established that H_2_O_2_ concentrations of 150 mM constitute the minimal inhibitory threshold for S. aureus growth suppression (Bucekova et al., 2023a). Despite considerable research investment, the mechanistic determinants underlying botanical origin-dependent variations in honey H_2_O_2_ accumulation remain incompletely characterized. Multiple physicochemical and biological parameters have been implicated in modulating H_2_O_2_ levels, including aqueous dilution, endogenous catalase activity, phytochemical composition, and post-harvest processing protocols and storage conditions. Emerging evidence indicates that honey H_2_O_2_ generation proceeds through an intricate biochemical network rather than a singular enzymatic pathway, with specific flavonoid compounds—including chrysin, apigenin, kaempferol, quercetin, and myricetin—demonstrating the capacity to attenuate GOX catalytic activity (Sanhueza and Fuentes, 2025), thereby introducing an additional layer of regulatory complexity.

The present research provides mechanistic insights into how honey's complex phytochemical matrix regulates enzymatic activity, which has implications for honey's antimicrobial properties and processing effects. This investigation revealed marked differential GOX activity across honey types, with blossom honeys exhibiting significantly distinct enzymatic activity profiles compared to mixed and honeydew varieties. Notably, TPPC exerted divergent modulatory effects on GOX activity in a botanical origin-dependent manner, demonstrating positive correlation in blossom honeys while exhibiting inverse correlation in honeydew honeys. This dichotomous response was particularly pronounced in honeydew samples, where escalating TPPC concentrations were associated with progressive attenuation of catalytic activity, whereas blossom honeys displayed the converse relationship. Quantitative analysis revealed that honey samples with TPPC concentrations spanning 40 to 50 mg GAE/100 g demonstrated optimal GOX enzymatic activity, suggesting a critical threshold range for maximal catalytic efficiency.

Conversely, semi-quantitative immunodetection of GOX protein abundance across honeys of diverse botanical provenance revealed no significant inter-sample variation in enzyme content, corroborating prior observations (Bucekova et al., 2019). The postulated mechanism by which honeybees differentially secrete elevated quantities of GOX enzyme during honeydew processing received no empirical support from our data, indicating that variations in enzymatic activity are attributable to post-secretion modulatory factors rather than differential enzyme deposition.

A key finding of this study is the demonstration that GOX enzymatic activity is significantly enhanced in multifloral and honeydew honey samples due to the presence of specific phytochemicals, predominantly polyphenolic compounds. Exposure to high-temperature thermal processing (100 °C) irreversibly compromised the structural integrity of both the GOX enzyme and associated phenolic and flavonoid constituents, resulting in complete ablation of enzymatic activity. Previous investigations have established that intensive thermal treatment induces marked depletion of phenolic compounds in floral honeys through polyphenol depolymerization and phenolic acid decarboxylation (Chaikham et al., 2016; Zarei et al., 2019). Notably, the magnitude of TPPC reduction and corresponding diminution in antioxidant capacity exhibits considerable heterogeneity across honey types, with botanical origin and environmental parameters representing critical determinants of these thermally induced alterations. On the other hand, the role of some cations present in honeys is probably important as GOX enzyme inhibitors, since they are not thermolabile and maintain their effect after heating the honey. These cations could form complexes with phytochemicals, making them unavailable. Thermal degradation of these ligands destroys the complexes. This represents an indirect mechanism of action of phytochemicals on GOX enzymatic activity (Sanhueza and Fuentes, 2025).

Given the thermolabile nature of both the GOX enzyme and honey polyphenols, dialysis was performed using cassettes with an MWCO of 3500 Da to isolate high-molecular-weight components. While substantial attenuation of GOX enzymatic activity was observed post-dialysis, quantitative analysis revealed significant depletion of GOX enzyme content in 4 of 6 dialysed samples, attributable to extended dialysis duration. Although suboptimal recovery of honey protein enzymes during dialysis has been documented in previous studies, this approach effectively eliminates low-molecular-weight compounds including organic acids, amino acids, minerals, vitamins, phenolics, and mineral salts (Akyıldız et al., 2022). Remarkably, both dialysed honeydew honey samples (Nos. 96 and 98) exhibited diminished enzymatic activity while maintaining unchanged relative GOX enzyme concentrations compared to their non-dialysed counterparts. This finding provides compelling evidence that specific phytochemicals function as enzymatic activity enhancers and may serve as non-protein cofactors possessing distinct chemical properties. Optimal catalytic function of GOX requires the non-covalent association of flavin adenine dinucleotide (FAD), which functions as the primary electron acceptor (Bauer et al., 2022). In addition, honey phytochemicals could form complexes with metals (e.g., Cu^2+^, Ag^+^) that inhibit GOX activity by interacting with the cofactor FAD, thereby favouring GOX activity at low concentrations under this mechanism but inhibiting it at higher concentrations. Paradoxically, while FAD has not been conclusively identified in honey matrices, certain honey varieties—particularly honeydew honeys—demonstrate substantially elevated H_2_O_2_ generation capacity relative to monofloral blossom honeys (Bucekova et al., 2018), suggesting the involvement of alternative catalytic mechanisms or cofactor systems.

Accumulating evidence indicates that polyphenolic autoxidation represents a distinct non-enzymatic pathway for H_2_O_2_ generation that operates independently of GOX-mediated catalysis (Brudzynski, 2020). This mechanism involves polyphenols functioning as electron donors in the reduction of molecular oxygen to superoxide radical anions (O_2_˙^-^), which subsequently dismutate to yield H_2_O_2_.

Despite constituting minor compositional fractions of honey, polyphenolic compounds—particularly flavonoids—exert disproportionate influence on the antimicrobial and anti-inflammatory bioactivity profiles of honey. The direct bactericidal efficacy of individual flavonoids, quantified through MIC determinations, spans a broad concentration range from 0.5 to >16,000 μg/mL (Li et al., 2025; Zhang et al., 2025), exceeding endogenous flavonoid concentrations in honey matrices by several orders of magnitude. Recent computational molecular docking analyses demonstrated that ten discrete polyphenolic constituents—including ferulic acid, quercetin, rosmarinic acid, and rutin—isolated from oak (Quercus spp.) and fir (Abies alba) honeydew honeys exhibited favorable binding interactions with seven bacterial virulence-associated protein targets (Hulea et al., 2025). Furthermore, in silico ADMET (absorption, distribution, metabolism, excretion, and toxicity) profiling revealed that beta-resorcylic acid, quercetin, ferulic acid, and p-coumaric acid demonstrated favorable therapeutic advantages. Consistent with this mechanistic framework, chrysin and ellagic acid isolated from Yemeni Sidr (Ziziphus spina-christi) honey were identified as principal bioactive constituents within secondary metabolite extracts, exhibiting antimicrobial efficacy through selective DNA gyrase inhibition, which compromises chromosomal topology maintenance and triggers downstream bacterial cytotoxicity (Alhadrami et al., 2025).

The comprehensive honey panel examined in this study, encompassing diverse botanical sources, enabled systematic characterization of GOX enzymatic activity, TPPC content, and antimicrobial efficacy against the model pathogen S. aureus across three distinct honey classifications: blossom honeys (monofloral and multifloral), mixed honeys (blossom-honeydew blends), and honeydew honeys. These bioactive parameters collectively govern the therapeutic potential of medical-grade honey formulations employed as wound care medical devices. Notably, despite widespread clinical adoption of medical-grade honey in wound management, standardized quality criteria and quantitative thresholds for antimicrobial and anti-inflammatory bioactivity remain unestablished in regulatory frameworks (Peters et al., 2025). Our findings suggest that TPPC concentrations and GOX enzymatic activity represent robust, quantifiable markers for rational selection of botanical sources in medical-grade honey production, potentially establishing evidence-based quality control parameters for therapeutic honey standardization.

Conclusions

5

This investigation of 99 honey samples reveals that GOX enzymatic activity and H_2_O_2_ generation are governed by phytochemical-enzyme interactions rather than enzyme abundance. The demonstration of botanical origin-dependent divergent effects—wherein polyphenols enhance GOX activity in blossom honeys yet attenuate activity in honeydew honeys—fundamentally challenges conventional assumptions of uniform enzymatic catalysis in honey matrices. The optimal TPPC range (40-50 mg GAE/100 g) for maximal activity indicates threshold-dependent regulation. Dialysis experiments provide definitive evidence that low-molecular-weight phytochemicals (<3500 Da) function as essential non-protein cofactors, potentially compensating for the absence of FAD or modulating enzyme conformation.

Complete activity abolishment following thermal processing (100 °C) and differential enhancement in proteinase K-treated honeydew samples (37-92% increase) demonstrate that thermolabile phytochemicals are indispensable for optimal catalytic function. From an applied perspective, TPPC concentration and GOX activity constitute synergistic quality control parameters for rational honey source selection in medical-grade formulations. The sensitivity of GOX-phytochemical interactions to thermal processing necessitates careful temperature control during honey standardization protocols. These mechanistic insights establish a foundation for evidence-based quality criteria for therapeutic honey applications and warrant further investigation of specific polyphenolic structures responsible for catalytic modulation.

CRediT authorship contribution statement

Marcela Bucekova: Conceptualization, data curation, formal analysis, investigation, methodology, writing—original draft, writing—review and editing. Jana Godocikova: Conceptualization, investigation, writing—review and editing. Simona Cigankova: Conceptualization, investigation, methodology, writing—review and editing. Viktoriia Chirkova: Investigation, writing—review and editing. Martin Safranek: Investigation, writing—review and editing. Juraj Majtan: Conceptualization, formal analysis, funding acquisition, project administration, supervision, writing—original draft, writing—review and editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.