# Screening of Some Romanian Raw Honeys and Their Probiotic Potential Evaluation
## 2 Materials and Methods
### Physico-Chemical Characterization of Raw Honey Samples  
#### 2.2.1 Nutritional Parameters of Raw Honey Samples  
The _melisospulynological analysis_ was made after the method of [PERSON] et al. (1978) [26], without acetolysis. Briefly, 10 g of honey was dissolved in diluted H\({}_{2}\)SO\({}_{4}\) (5%), centrifugal 10 min (3396 rcf), and the sediment washed twice with distilled water. After another centrifugation, the supernatant was discharged, and two drops of liquid, together with the entire sediment, was put on a microscopic slide, and an area 20 \(\times\) 20 m\({}^{2}\) was mounted with glycerine-gelatine-fuxine. Slide examination was performed using an Olympus BX51 microscope at 1000\(\times\)magnification (for identification) and 400\(\times\)magnification for counting. One thousand pollen grains were counted from every slide, and percentages of different botanical species were calculated. For the identification of pollen type, different pollen guides were used. Figure 1: Geographical spread of raw honey samples (P1–P10). Moisture content:The method used for moisture content was adapted from International Honey Commission (IHC) [27], measuring the refractive index of a liquid honey sample, using Abbe refractometer (Abbe WAY-S Selecta--Spain). In the case of a crystalized sample, honey was placed in a tube with rod and was heated in a water bath at 40 \({}^{\circ}\)C until liquification. The refractive index read at 20 \({}^{\circ}\)C on the refractometer is transformed in water content using the table from the standard. Results are expressed as % water. Sugars profile determination:HPLC (High-performance liquid chromatography) analysis of the carbohydrates is carried out on a modified Alltima Amino 100 A stainless steel column (Alltech, Nicholasville, KY, USA) (4.6 mm diameter, 250 mm length, particle size 5 \(\upmu\)m) following IHC method modified in APHIS-DIA Laboratory [28]. The SHIMADZU instrument (LC-10 AD VP model, Shimadzu, Kyoto, Japan) was equipped with degasser, two pumps, auto sampler, thermostat oven, controller, and refractive index detector. The injection volume was 10 \(\upmu\)L, and the flow rate was 1.3 mL/min. The mobile phase was acetonitrile/water (75:25 \(v/v\)). Briefly, 5 g of honey were dissolved in ultrapure water (40 mL), transferred into a 50 mL volumetric flask, containing 25 mL HPLC grade purity methanol, and subsequently filled to the mark with water. The honey solution was filtered through a 0.45 \(\upmu\)m membrane filter, collected in HPLC glass vials for analysis. A calibration curve for each sugar was made using standard solutions of different concentrations (0.5-80 mg/mL) for the quantification of sugars, with regression coefficients r\({}^{2}\) higher than 0.998. Quantification was made by comparing the obtained peak area with those of standard sugars. The results were expressed as g/100 g honey. Total lipids:The content of total lipids in honey samples was determined using Soxhlet method ([PERSON], Gerhardt, Germany), a literature method [29] modified in APHIS-DIA Laboratory. Briefly, 2 g of honey samples were weighted on filter paper, and also the extraction glasses containing 2 boiling stones, will be weighted and, after that, together with the cartridge and the solvent (90 mL etil-eteric), will be fixed in PTFE cylinders. The method is set from the multistat: extraction temperature 140 \({}^{\circ}\)C, extraction time 5 h, washing 30 min, solvent evaporation in hot air flow 10 min. After extraction ends, extraction glasses will be placed in the oven at 60 \({}^{\circ}\)C, for one half hour, to eliminate traces of solvent and, after cooling, will be weighted, and the results were expressed as percent. Total proteins:The content of total proteins of honey samples was determined using Bradford method. The original method of Bradford [30] was modified in our laboratory, for honey analysis. Briefly, 2 g of honey samples dissolve with 2 mL of ultrapure water in a 10 mL Berzelius glass.