2.6. Determination of HMF and Diastase Levels

HMF and diastase levels were determined in each honey sample following the Harmonised Methods of the International Honey Commission [17]. More specifically, for the quantification of HMF (mg/kg), the spectrophotometric method of White [18] was applied. To do this, 5 g of each honey sample was diluted in 25 mL of water and transferred into a 50 mL volumetric flask. Then, 0.5 mL of Carrez I solution was added, followed by 0.5 mL of Carrez II solution in the slash, which was filled up to 50 mL with water. The solution was filtered through paper and the first 10 mL of the filtrate was rejected. Aliquots of 5 mL were added to two test tubes, and 5 mL of distilled water was added to one tube (sample solution), while 5 mL of 0.2% (w/v) sodium bisulphite solution was added to the second (reference solution). The absorbances of the solutions at 284 and 336 nm were determined using a UV–visible spectrometer (UV-1800, Shimadzu Co., Kyoto, Japan). The HMF content was calculated by the equation HMF (mg/kg) = A₂₈₄ − A_{336 ×} 149.7 where A₂₈₄ is the absorbance at 284 nm, A₃₃₆ is the absorbance at 336 nm and 149.7 is a factor calculated by the molecular weight of HMF and the mass of the sample.

For the quantification of diastase, the methodology initially described by Schade et al. [19] was followed. To do this, 10 g of each honey sample was placed in a 50 mL beaker in which 5 mL of acetate buffer was added together with 20 mL of water. After samples were dissolved, 3 mL of sodium chloride (0.5 M) were added, and the solution was diluted to 50 mL with water. At the same time, a starch solution was standardized using an iodine solution. Both solutions were warmed at 40 °C. Starch solution (5 mL) was added into 10 mL of honey solution and a stopwatch was started. An aliquot was taken every 5 min and added to 10 mL of iodine solution. The absorbance was recorded and a calibration curve was obtained. According to the Association of Official Analytical Chemists (AOAC) method, the number 300 was divided by the time needed to reach the absorbance value of 0.235 and expressed as Diastase Number (DN) [20]. All chemicals used for these experiments were purchased from Merck KGaA (Darmstadt, Germany).