# Screening of Some Romanian Raw Honeys and Their Probiotic Potential Evaluation
## 2 Materials and Methods
### Physico-Chemical Characterization of Raw Honey Samples
#### 2.2.4 Quality Analysis of Raw Honey Samples  
_Hydroxymethylfurfural content_: The concentration of hydroxymethylfurfural (5-hydroxy methill-furan-2-carbableide--HMF) content was determined via high performance liquid chromatography with photodiode array detection (HPLC-PDA) [27] (Shimadzu VP system--Japan). Operational parameters of chromatographic system: mobile phase ultrapure water (solvent A), methanol (solvent B), gradient scheme: 10% B at 0-13 min, 90% B at 13-15 min, 90% B at 20 min, and 90-10% B at 21 min, equilibrating the column (10% B) until 30 min, flow rate 0.8 mL/min, injection volume 20 \(\mathrm{\SIUnitSymbolMicro L}\), Discovery HS C18, 5 \(\mathrm{\SIUnitSymbolMicro m}\) 4.6 \(\times\) 250 mm column, column temperature 30 \({}^{\circ}\)C, detection at 283 nm. Ten grams of honey is weighted into a Berzelius glass and dissolved in approximately 25 mL of distilled water. Honey solution is transferred quantitatively into a 50 mL volumetric flask. Add 0.5 mL of Carrez I solution (15 g of potassium hexacyanoferrate (II), K4 Fe(CN)6 \(\times\) 3H2O in water and make up to the mark in 100 mL) and mix. Then, add 0.5 mL of Carrez II solution (30 g of zinc acetate, Zn(CH3 COO)2 \(\times\) 2H2O, and make up to 100 mL), mix, and make up to 100 mL with water. Filter through filter paper, removing the first 10 mL. The filtrate was afterwards filtered into glass vial through a 0.45 \(\mathrm{\SIUnitSymbolMicro m}\) syringe filter and inject into HPLC system. Different concentrations of HMF (Hidroximetilfurfural) standard (1-50 mg/mL) were injected into the chromatographic system and a calibration curve was constructed. The concentration of HMF from the samples is calculated with the equation:  
\[HMF(mg/kg)=\frac{\mathrm{Cc}\times 5\times 10}{m},\]  
where:  
Cc--concentration from the calibration curve,  
_m_--mass of weighted honey, and  
results are expressed in mg/kg honey. _Diastase activity_: For the determination of diastazic activity, Amylazine--Megazyme spectrophotometric method was used [33]. Amylazine tablets contain amylose (the linear fraction of starch). In the presence of \(\alpha\)-amylase (from the honey solution), the substrate is hydrolyzed, and the soluble colored product is released. The reaction is terminated by the addition of Trizma solution, followed by filtration. The absorbance of the filtrate is read at 590 nm towards a control sample (without honey) as blank. There is a linear correlation between absorbance of the filtrate and the diastase activity in the sample. For sample preparation, 2 g of honey was weighted and dissolved in 40 mL of 100 mM maleate buffer (pH 5.6) in a 50 mL Berzelius glass. The solution was then transferred quantitatively into a 50 mL volumetric flask and make up to the mark with water. One mL of the diluted honey solution was placed in a test tube and pre-incubated at 40 \({}^{\circ}\)C for 5 min. One tablet of Amylazine was added to the tube without removing the test tube from the water bath. The tablet hydrated quickly and absorbed most of the liquid. The samples were then incubated the tube at 40 \({}^{\circ}\)C for exactly 10 min; afterwards, 10 mL of basic Trizma solution (2% \(w/v\)) was added to complete the reaction, and the test tube was shacked vigorously and stored at room temperature for 5 min. The content of the tubes was filtered through paper filter and read at 590 nm on a spectrophotometer (UV-1700 Shimadzu Instruments) against a control. Diastazic activity was calculated by the following formula: 20 \(\times\) DO\({}_{590}\), where DO\({}_{590}\) represents the absorbance. Results are expressed in Schade units/g honey. _Contaminants: tetracycline and oxytetracycline:_ Tetracycline and oxytetracycline from honey were determined using high performance liquid chromatography with photodiode array detection (HPLC-PDA). The method was adapted from AOAC methods [34] in our laboratory for the detection and quantification of antibiotics from honey.