Toward the ultimate assay for honey authentication: combining multidimensional DNA sequencing data with multi-platform physical-chemical and isotopic d13C analyses

One of the most pressing issues facing the global apicultural sector is the frequent presence of honey on the market that does not meet regulatory requirements. This is evidenced by the numerous notifications of adulterated honey. The most common form of adulteration involves extending honey with sugar syrups. This malpractice presents enticing opportunities for fraudulent business operators due to the low cost of sugar syrup and the challenges associated with current analytical approaches in detecting them in honey. Other frauds include mislabeling the geographic, botanical and entomological origins of honey. Given the complexity of the issue and the extensive information required to detect adulterated honey and frauds in this production system, it is necessary to utilise a combination of analytical approaches and methodologies capable of examining different components and chemical-physical parameters of honey and foreign substances. In this study, we utilised a combination of various methodologies to analyse authentic, suspected and adulterated honey samples from different countries with various declared botanical origins. These methodologies included: 1) nextgeneration DNA sequencing techniques involving five plant and entomological metabarcoding analyses and whole DNA shot-gun sequencing, with advanced bioinformatic approaches for interpreting sequencing data and identifying multiple environmental DNA fingerprints in honey; 2) melissopalynological analysis to identify the pollen spectrum present in the samples; 3) standard chemical-physical parameters, such as humidity, acidity, hydroxymethylfurfural, diastase and pH; 4) standardized isotopic-based methods, including Elemental Analyzer (EA) for determining C-4 plant sugar in honey and Liquid Chromatography (LC) Coupled to Isotope Ratio Mass Spectrometry (LC-IRMS) method for determining the δ13C ratios of mono-, di-, and trisaccharides in honey; 5) Liquid Chromatography High Resolution MS (LC-HRMS) in target mode to detect markers of the sugar syrups. The combination of results from these different methodologies provided a wealth of information that allowed us to effectively group honey samples in authentic or adulterated. Our pilot test suggests that using multiple methods is necessary to identify adulterated honey samples. We are working on constructing a reference database with multiple analytical information to further enhance our ability to identify counterfeit honey.