NMR-Based Chemical Profiling and Chemometric Analysis of Brazilian Stingless Bee Honeys

Brazil harbors a remarkable diversity of native stingless bees, whose honeys exhibit distinctive chemical compositions. Despite the long-standing traditional knowledge associated with meliponiculture, information on the chemical diversity of stingless bee honeys remains limited. Nuclear magnetic resonance (NMR) spectroscopy provides comprehensive chemical fingerprints of honey with minimal sample preparation and has emerged as a valuable tool for investigating compositional variability among honey samples. When combined with multivariate analysis, NMR-based profiling can reveal chemical patterns associated with different sample groups and support their discrimination. This study aimed to obtain NMR-based chemical profiles of honeys produced by Brazilian stingless bees and investigate differences among bee taxa. Thirteen honey samples representing three genera were analyzed by NMR spectroscopy. One-dimensional (1D) ^1H NMR and two-dimensional HSQC and TOCSY spectra were acquired using a 500 MHz NMR spectrometer. Metabolite assignments were supported by COLMAR-assisted analysis of 1D and 2D NMR spectra and comparison with spectral data. The ^1H NMR spectra were processed by peak-based binning, and chemometric analyses, including principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), were performed using MetaboAnalyst 6.0. Distinct spectral patterns were observed among the analyzed taxa, particularly in carbohydrate-associated regions. Spectra from Duckeola, Oxytrigona, and Tetragona honeys exhibited a signal at approximately δ 4.9 ppm. Honeys from Tetragona clavipes and Duckeola ghilianii showed lower intensities for signals at approximately δ 4.6 and 5.2 ppm, the latter assigned to glucose, while T. clavipes displayed a distinct spectral profile within the carbohydrate-rich region between δ 3.20 and 4.15 ppm. Multivariate analysis further revealed differences in chemical profiles among the samples. PCA and PLS-DA showed clearer separation among the non-Melipona genera, whereas Melipona samples exhibited greater overlap. Within Melipona, M. flavolineata and M. mondury showed greater differentiation from the remaining samples. Preliminary evaluation of HSQC and TOCSY spectra indicated the presence of mannitol in M. mondury, M. quadrifasciata anthidioides, M. scutellaris, M. flavolineata, M. seminigra, and T. clavipes. Mannitol has recently been proposed as a potential chemical marker of stingless bee honey; however, under the analytical conditions employed in the present study, it was not detected in all samples. This finding suggests that its detectability by NMR may vary among stingless bee honeys and warrants further investigation before its broader application as a universal chemical marker. Overall, NMR-based chemical profiling revealed compositional variability among Brazilian stingless bee honeys and demonstrated its potential for identifying taxon-associated chemical patterns. 

Acknowledgment: CNPq and FAPERJ.