Przemysław Leszczyński, Dorota Wojnicz, Dorota Tichaczek-Goska, Michał Gleńsk, Alan Gasiński, Joanna Kawa-Rygielska
Optimizing hop (Humulus lupulus L.) extraction protocols for maximum antibacterial efficacy remains an open methodological challenge in developing plant-based natural antimicrobials. We compared five extraction protocols-hot ethanol, cold ethanol, espresso/steam, hot water, and cold water-applied to four hop varieties (Citra, Lubelski, Magnat and Marynka), with a commercial supercritical CO2 extract as a reference preparation. We characterized extracts by HPLC (α-acids, β-acids, iso-α-acids) and GC-MS (volatile profile of cold ethanol extracts) and determined antibacterial activity as minimum inhibitory concentration (MIC) against reference and clinically relevant multidrug-resistant (MDR) strains of Escherichia coli and Staphylococcus aureus. Friedman tests confirmed a statistically significant effect of extraction method on MIC values (p ≤ 0.004 for E. coli ATCC 25922, S. aureus ATCC 29213, and clinical MRSA). Ethanol-based protocols consistently produced the highest hop acid concentrations and the lowest MICs against S. aureus ATCC 29213 (0.125-0.250 mg/mL) and against clinical MRSA (as low as 0.016 mg/mL), compared with 1.0-15.0 mg/mL for aqueous and steam-based extracts. Clinical MRSA demonstrated increased susceptibility to selected ethanol extracts and to the CO2 reference. MDR uropathogenic E. coli (UPEC) maintained high-level resistance (MIC 15 mg/mL) to all preparations except the CO2 reference (MIC 6 mg/mL). Spearman correlation identified α-acid content as the dominant predictor of antibacterial activity (ρ = -0.927, p < 0.001 for S. aureus ATCC 29213). These results suggest ethanol-based and supercritical CO2 extraction as optimal protocols for producing hop extracts with antibacterial properties targeting Gram-positive pathogens, including MRSA, and provide a rationale for their use as natural antimicrobials.