## Materials and Methods
### Determination of the Antibacterial Activity
#### 2.2.2 Study Design  
Used Solvents  
In the present study, as a solvent to prepare various solutions and concentrations of honey, phosphate-buffered saline (PBS) was used. A simulation of in vivo (oral cavity) testing was attempted and all the tests for the determination of the minimum inhibitory concentration (MIC), with the difference that artificial saliva also was used as solvent of the honey samples. The artificial saliva was prepared according to the composition used in dental studies, as shown in Table 1[52; 53]. The mucin used in this study was porcine gastric mucin, which comprises of both human mucins type MUC5 AC and type MUC6 [54] and is said to simulate human saliva rymucins (mucin type MUC5B) [55]. Introduction on the Methods of Assessment of Antimicrobial Potency of the Different Honey Types  
For the assessment of the antibacterial potency of the different types of honey, two different in vitro methods were used: (i) Agar wells diffusion method, and (ii) determination of the minimum inhibitory concentration by microtiter plates. The first method is based on the inhibition of bacterial growth in a circular zone around the well. The second method is based on the inhibition of bacterial growth in different dilutions of honey. In order to investigate the possible modes of antibacterial action involved, four different techniques of MIC determination by microtiter plates were used: (a1) addition of catalase, with PBS as solvent (a2) addition of catalase, with artificial saliva as solvent, (b1) addition of proteinase K, with PBS as solvent and (b2) addition of proteinase K, with artificial saliva as solvent. It should be noted that the agar well diffusion assay was performed only for the 50% dilution of honey to simulate the dilution of honey in the oral cavity. It is estimated that a spoon of honey was screened for its antibacterial activity, according to the agar well diffusion method proposed by the Clinical and Laboratory Standards Institute (CLSI, former NCCLS) guidelines [20]. Briefly, overnight bacterial cultures grown in Mueller-Hinton broth were adjusted to 0.5 McFarland turbidity standard (\(\sim\)1.5 \(\times\) 10\({}^{8}\) CFU/mL). Mueller-Hinton agar plates were inoculated with roughly 10\({}^{6}\) CFUs over the entire surface of the plate. Wells of 8 mm in diameter were cut into the surface of the agar using a sterile cork borer. Subsequently, 100 \(\mu\)L (50% \(v/v\) in phosphate-buffered saline (PBS)) of the tested honey types, manuka honey, and artificial honey were added separately to each well. The plates were incubated aerobically and anaerobically at 37 \({}^{\circ}\)C for 16-18 h. Antibacterial activity was assessed by measuring (with a calliper) the diameter of the inhibition zones surrounding the wells, including the diameter of the well. The diameter of the inhibition zone, if present in the negative control, was recorded and subtracted from the inhibition zones of the tested honey, as well as of manuka honey. The experiment was repeated three times. Determination of Minimum Inhibitory Concentration (MIC)  
The minimum inhibitory concentration (MIC) of the honey types was determined in sterile 96-well polystyrene microtiter plates (Kisker Biotech GmbH and Co. KG, Steinfurt, Germany) by using a spectrophotometric bioassay, as previously described [20,57]. Briefly, overnight bacterial cultures grown in Mueller-Hinton broth were adjusted to a 0.5 McFarland turbidity standard (\(\sim\)1.5 \(\times\) 10\({}^{8}\) CFU/mL). Approximately 5 \(\times\) 10\({}^{4}\) CFUs in 10 \(\mu\)Mueller-Hinton broth were added to 190 \(\mu\)L of 2-fold diluted test honey (honey concentration ranged from 75 to 0.58% \(w/v\)) in Mueller-Hinton broth. Two-fold serial dilutions of the same range of manuka honey and artificial honey were included for comparison. The control wells contained only Mueller-Hinton broth-inoculated with bacteria.