The minimum inhibitory concentration (MIC) of increasing concentration of each bee pollen samples (1, 2.5, 5, and 10 mg/mL) was determined according to Frassinetti et al. [15] on selected pathogenic bacteria, mainly three Gram-negative and two Gram-positive strains. Pathogenic microorganisms were maintained for 16 h at 37 °C in MHB; then, cultures were diluted to match the 0.5 McFarland standard turbidity. A typical mixture contained 50 µL of bacterial suspensions, corresponding to about 1–5 × 105 CFU/mL MHB, 100 µL of bee pollen extract dilutions, and 100 µL of MHB in transparent sterile 96-well plates. Each plate was maintained at 37 °C for 24 h in aerobic conditions and a control, containing only the bacterial inoculum in MHB, was included on each one. Gentamicin and vancomycin were used as positive control (1 mg/mL in sterile physiological solution, corresponding to 0.05 mg/mL in the well). The optical density (O.D.) values were recorded at 600 nm, and the lowest concentration of bee pollen extracts able to suppress the microorganisms’ growth was defined as the MIC value.

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