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

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.