This novel review of analytical methods for pot-honey research was intended to provide concise references to a 35-day post-harvest experiments at 30 °C, in an integrated study. Diverse methods were selected from specialized literature, from the AOAC (Association of Official Analytical Chemists), and the International Honey Commission. Besides the geographical and seasonal origin, the pot-honey I.D. consists of entomological and botanical identifications, the latter performed by acetolyzed or natural melissopalynology. The methods of this integrative study included: 1. Physicochemical analysis (Aw, color, moisture, pH, free acidity, lactone acidity, total acidity, hydroxymethylfurfural (HMF), and sugars by highperformance liquid chromatography HPLC), 2. Targeted proton nuclear magnetic resonance 1H-NMR metabolomics (sugars, ethanol, HMF, aliphatic organic acids, amino acids, and botanical markers), 3. Biochemical composition (flavonoids, polyphenols), 4. Antioxidant activity (ABTS 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid-free radical scavenging assay, DPPH 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay, ferric reduction assay FRAP), 5. Microbial counts (aerobic plate, yeast and mold, Bacillus, and lactic acid bacteria count), 6. Honey microbiome profiling via independent-culture method: high-throughput bacteria and fungi based on amplicon sequencing approaches, 7. Sensory evaluation (odor, aroma, taste, persistence), and 8. Honey authenticity and biosurfactant tests by an interphase emulsion. A further section was included to provide basic information on the results obtained using each method. This was needed to explain the interacting components derived from pot-honey processing within the stingless bee nest and post-harvest transformations.

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The biodiversity of Ecuadorian stingless bees is almost 200 species. Traditional pot-honey harvest in Ecuador is mostly done from nests of the three genera selected here Geotrigona Moure, 1942, Melipona, Illiger, 1806 and Scaptotrigona, Moure 1943. The 20 pot-honey samples collected from cerumen pots and three ethnic honeys “abeja de tierra”, “bermejo”, and “cushillomishki” were analyzed for qualitative and quantitative 1H-NMR honey profiling and for the Honey Authenticity Test by Interphase Emulsion (HATIE). Extensive data of targeted organic compounds (41 parameters) was identified, quantified and described. The three honey types were compared by ANOVA. Amino acids, ethanol, hydroxymethylfurfural, aliphatic organic acids, sugars, and markers of botanical or entomological origin. The number of phases observed with the HATIE was one in Scaptotrigona and three in Geotrigona and Melipona honeys. Acetic acid (19.60 ± 1.45 g/kg) and lactic acid (24.30 ± 1.65 g/kg) were particularly high in Geotrigona honey (in contrast to 1.3 g/kg acetic acid and 1.6 g/kg lactic acid in Melipona and Scaptotrigona), with the lowest fructose + glucose (18.39 ± 1.68) g/100g honey compared to Melipona (52.87 ± 1.75) and Scaptotrigona (52.17 ± 0.60). Three local honeys were tested using PCA (Principal Component Analysis), two were assigned with a correct declared bee origin, but “bermejo” was not a Melipona and grouped with the Scaptotrigona cluster. However, after HCA (Hierarchical Cluster Analysis), the three kinds of honey were positioned in the Melipona-Scaptotrigona cluster. This research supports the targeted NMR-based profiling in pot-honey metabolomics approach for multi-parameter visualization of organic compounds, descriptive and pertained multivariate statistics (Hierarchical Cluster Analysis HCA, and Principal Component Analysis PCA) to discriminate the stingless bee genus in a set of Geotrigona, Melipona and Scaptotrigona honey types. The NMR characterization of Ecuadorian honey produced by stingless bees is a contribution to the needed regulatory norms. A final note on searching stingless bee markers in pot-honey metabolites that may become nutritional trait candidates for phylogeny. Scaptotrigona honey revealed biosurfactant activity in the HATIE, originating a fingerprint Honey Biosurfactant Test (HBT) for the genus in this set of pot-honeys.

This novel review of analytical methods for pot-honey research was intended to provide concise references to a 35-day post-harvest experiments at 30 °C, in an integrated study. Diverse methods were selected from specialized literature, from the AOAC (Association of Official Analytical Chemists), and the International Honey Commission. Besides the geographical and seasonal origin, the pot-honey I.D. consists of entomological and botanical identifications, the latter performed by acetolyzed or natural melissopalynology. The methods of this integrative study included: 1. Physicochemical analysis (Aw, color, moisture, pH, free acidity, lactone acidity, total acidity, hydroxymethylfurfural (HMF), and sugars by highperformance liquid chromatography HPLC), 2. Targeted proton nuclear magnetic resonance 1H-NMR metabolomics (sugars, ethanol, HMF, aliphatic organic acids, amino acids, and botanical markers), 3. Biochemical composition (flavonoids, polyphenols), 4. Antioxidant activity (ABTS 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid-free radical scavenging assay, DPPH 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay, ferric reduction assay FRAP), 5. Microbial counts (aerobic plate, yeast and mold, Bacillus, and lactic acid bacteria count), 6. Honey microbiome profiling via independent-culture method: high-throughput bacteria and fungi based on amplicon sequencing approaches, 7. Sensory evaluation (odor, aroma, taste, persistence), and 8. Honey authenticity and biosurfactant tests by an interphase emulsion. A further section was included to provide basic information on the results obtained using each method. This was needed to explain the interacting components derived from pot-honey processing within the stingless bee nest and post-harvest transformations.