## Materials and Methods
### Determination of the Antibacterial Activity
#### 2.2.2 Study Design
The optical density (OD) was determined at 630 nm using a microplate reader (Multi-detection reader, BioTek(r)); just prior to incubation (t = 0) and after 24 h of incubation (t = 24) at 37 \({}^{\circ}\)C. MIC was determined as the lowest honey concentration that results in 100% growth inhibition. Determination of Minimum Inhibitory Concentration (MIC) Performed After Enzymatic Treatment of Honey Samples with Catalase and Proteinase K  
This determination was done following two options:  
1. by using catalase, which degrades hydrogen peroxide, allowing evaluation of the contribution of hydrogen peroxide production to antibacterial activity [58]. \begin{table}
\begin{tabular}{c c} \hline \hline \multicolumn{2}{c}{**Components in Artificial Saliva**} & **Concentration (g/L)** \\ \hline Sodium chloride & NaCl & 1.594 \\ Ammonium nitrate & NH\({}_{4}\)NO\({}_{3}\) & 0.328 \\ Potassium phosphate & KH\({}_{2}\)PO\({}_{4}\) & 0.636 \\ Potassium chloride & KCl & 0.202 \\ Potassium citrate & K\({}_{3}\)C\({}_{6}\)H\({}_{5}\)O\({}_{7}\): H\({}_{2}\)O & 0.308 \\ Uric acid sodium salt & C\({}_{6}\)H\({}_{3}\)N\({}_{4}\)O\({}_{3}\)Na & 0.021 \\ Urea & H\({}_{2}\)NCONH\({}_{2}\) & 0.198 \\ Lactic acid sodium salt & C\({}_{3}\)H\({}_{5}\)O\({}_{3}\)Na & 0.146 \\ Porcine gastric Mucin Type II & & 1.35 \\ D- (+) glucose & & 0.1 \\ \(\alpha\)-amylase & & 100,000 U \\ Lysozyme & & 750 U \\ Water & Four times distilled H\({}_{2}\)O and 0.1 M NaOH used to achieve pH 6.8 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Chemical composition of artificial saliva. Reprinted with permission from Refs. [55,56] 2009 Elsevier. 2. by adding proteinase K, with which the part of antibacterial activity that is due to proteins and peptides in honey can be assessed [59]. Catalase stock solution was prepared by dilution of 30 mg powder of catalase originating from bovine liver (SERNA, Heidelberg, Germany) to 10 mL phosphate buffer (pH 7.4. In 1.5 mL honey 50% \(v/v\) (750 \(\upmu\)L honey and 750 \(\upmu\)Muller Hinton Broth), 28 \(\upmu\)L of the stock solution were added so that the final concentration of catalase was 600 U/mL. The dilution of honey was put in the incubator shaker for 16 h at 37 \({}^{\circ}\)C in 210 rounds. Subsequently, all the different honey solutions were prepared to determine the MIC. For the proteinase K stock solution of 10 mg/mL concentration, 10 mg of proteinase powder (Ambion(r), Inc., Huntingdon, Cambridgeshire, UK) were diluted in 1 mL distilled water. The same procedure was followed as described for catalase to obtain the different dilutions, so that the final concentration of proteinase Kwas 100 \(\upmu\)g/mL. Catalase- and proteinase K-treated honey samples were then used in the antibacterial assay to determine the MIC values, as described in paragraph \"Determination of Minimum Inhibitory Concentration (MIC)\". Controls containing no honey (positive growth control), and no honey with catalase or proteinase K (catalase only control/ proteinase K only control) were included to evaluate the effect of catalase/proteinase K alone on bacterial growth. The elevated MIC values of the treated honey compared to the untreated honey revealed the presence of hydrogen peroxide and/or proteinaceous compounds, which contributed to the antibacterial activity of the tested honey types. Three-fold samples for every honey and for every concentration were examined. The physicochemical characteristics of honeys were expressed as means (\(\pm\)SD) of triplicate analyses. Results from the well diffusion assays and minimum inhibitory concentration are also presented as means (\(\pm\)SD) of mm or honey concentration (%). Analysis of variance with [PERSON]'s post-hoc comparison was used to compare either the physicochemical characteristics of the samples or the antibacterial effects. [PERSON]'s rho correlation coefficient was used to indicate any correlation between the physicochemical characteristics and cluster analysis to distinguish groups of observations with similar characteristics.