**2.14. Antimicrobial activity**

into a second tube and 0.5 ml of proline standard solution into a third tube were pipetted. 1 ml of formic acid and 1 ml of ninhydrin solution were added to each tube. After 15 min shaking, the tubes were incubated at 70°C for 10 min. 5ml of the 2-propanol-water-solution were added to each tube and the absorbance read at 510 nm after 45 min. The proline content in mg/kg honey

The respective standards and honey samples were prepared as 1% solution prior to analysis. Each standard and sample (2 μl) was injected in triplicates in a Dionex Thermo Fisher Ultra High Performance Liquid Chromatography with a charged aerosol detector, equipped with an amino column from Supelco (250 × 4.6 mm, 5 μm particles). Mobile phase consisted of a water/acetonitrile mixture (volume ratio 75/25), with a flow rate of 1.5 mL/min. Detection was performed at a Data Collection Rate of 20 Hz, filtered at 5 s, peak width was 0.02 mm and

The total phenolic content (TP) was determined using a Folin Ciocalteu test [9]. 100 μl of Folin-Ciocalteu reagent and 80 μl of sodium carbonate (1 M) were added to 10 μl of each honey stock solution (triplicates) and incubated for 20 min at room temperature. The absor-

The total flavonoid (TF) content was determined by a spectrophotometric method [10, 11]. Honey samples were prepared as 50% (w/v) solutions. 25 μl of honey solutions and 100 μl

NaOH solution and 25 μl of distilled water were added for a final volume of 250 μl. After 15 min, the absorbance was read at 510 nm. Rutin (Sigma-Aldrich, USA) was used as a standard

The DPPH assay (2,2-diphenyl-1-picrylhydrazyl, Sigma Aldrich, USA) was carried out according to Moien et al. [10]. Honey samples were prepared as 12.5% (w/v) solutions. 200 μl of a 100 μM solution of DPPH radical in methanol were added to 20 μl of honey solution, and incubated for 30 min in the dark at room temperature. The radical inhibition was measured at 490 nm against a blank containing DPPH and methanol. Ascorbic acid (BDH, UK) was used as a standard (10–100 μg/mL). The AAE-DPPH and ascorbic acid equivalence (mg AEAC/100

The FRAP (Ferric Reducing Antioxidant Power) activity of honey samples was determined according to Oyaizu [13]. 250 μl of honey solutions (6.25–50%) or 250 μl of distilled water

solution (Fisher Scientific, UK) were allowed to mix for 6 min. 100 μl of 4%

= 0.9962) [12].

bance was read at 630 nm. Tannic acid was used as a standard for the test.

for the quantification of the total flavonoid content (0–500 mg/mL; *r*<sup>2</sup>

**2.12. Radical scavenging activity: the DPPH assay**

= 0.9928) were calculated [12].

**2.13. Reducing power: the FRAP assay**

was calculated.

176 Honey Analysis

**2.9. Determination of sugar content**

oven temperature set to 35°C.

**2.10. Polyphenolic content**

**2.11. Total flavonoid content**

of 0.15% NaNO2

g honey) (*r*<sup>2</sup>

Maltese honey was tested against *Escherichia coli* ATCC® 25922, *Staphylococcus aureus* ATCC® 259213 and *Pseudomonas aeruginosa* ATCC® 27853. The medium of choice for broth dilution testing was cation-adjusted MHB (CAMHB). The activity was compared against artificial honey so as to show whether the activity is merely the result of the high osmotic potential of honey. This was prepared by dissolving 81 g D-fructose, 67 g D-glucose, 15 g maltose and 3 g sucrose in 34 ml filter sterilised distilled water [15]. A broth macro-dilution assay was used to analyse the honey and control samples [16]. 1 ml of each honey sample and artificial honey (0.0625–1.0 g/ml) and 1 ml of inoculum suspension (5 × 105 CFU/ml) were incubated for 20 hours at 35°C. The minimum inhibitory concentration (in g/ml) was then obtained.
