Assessment of the botanical origin of Bulgarian honey samples using melissopalynological, DNA barcoding and NMR analyses

Polyfloral honey samples from Bulgaria were subject to parallel analyses of their botanical origin and composition using traditional melissopalynology, DNAbarcoding based on the plastid rbcL gene and NMR analysis. The obtained datasets were compared with each other to evaluate the information capacity of the applied experimental methods. The results from the melissopalynological and DNA-barcoding studies demonstrated a significantly higher resolution of the latter, revealing the presence of pollen from a total of 17 plant families, 21 plant genera and 5 plant species in comparison to pollen from only 7 plant families, 3 plant genera and 4 plant species identified by melissopalynology. The higher resolution of DNA barcoding allows a more detailed characterisation of the diet and foraging preferences of honey bees, including foraging on plant species growing in lower abundance in the area. The comparison of the quantitative data on floral honey composition for several plant genera and species reveals significant differences between the relative abundance of the pollen grains estimated by melissopalynological analysis and the relative abundance of rbcL clones in rbcL libraries determined after DNA barcoding. All three applied methods confirm the polyfloral botanical origin of the analysed samples and support routine NMR use for the assessment of the floral origin of honey

Optical Rotation—A Reliable Parameter for Authentication of Honey?

The controversial question of whether optical rotation data can be used to distinguish floral from honeydew honey was investigated. Specific optical rotation angles were determined for 41 honey samples, including floral, honeydew, and adulterated honey, indicating that moderate to high positive optical rotation angles were found for all adulterated samples measured. A strong correlation between the sugar profile and the specific optical rotation angle of honey was confirmed, and a method based on 13C NMR metabolomics was proposed to calculate specific optical rotation angles with good correlation with the experimental values. The results indicate that optical rotation is not a reliable method for distinguishing the origin of honey but could indicate adulteration

NMR Profiling of North Macedonian and Bulgarian Honeys for Detection of Botanical and Geographical Origin

Bulgaria and North Macedonia have a long history of the production and use of honey; however, there is an obvious lack of systematic and in-depth research on honey from both countries. The oak honeydew honey is of particular interest, as it is highly valued by consumers because of its health benefits. The aim of this study was to characterize honeydew and floral honeys from Bulgaria and North Macedonia based on their NMR profiles. The 1D and 2D 1H and 13C-NMR spectra were measured of 16 North Macedonian and 22 Bulgarian honey samples. A total of 25 individual substances were identified, including quinovose, which was found for the first time in honey. Chemometric methods (PCA—principal component analysis, PLS-DA—partial least squares discriminant analysis, ANOVA—analysis of variance) were used to detect similarities and differences between samples, as well as to determine their botanical and geographical origin. Semiquantitative data on individual sugars and some other constituents were obtained, which allowed for the reliable classification of honey samples by botanical and geographical origin, based on chemometric approaches. The results enabled us to distinguish oak honeydew honey from other honey types, and to determine the country of origin. NMR was a rapid and convenient method, avoiding the need for other more time-consuming analytical techniques