Chemical fingerprint of phenolamides and flavonoid glycosides in bee pollen and bee bread from different altitudes and botanical origins

In this study, we characterised and compared the metabolites of multifloral samples of bee pollen (BP) and bee bread (BB) collected from two A. mellifera ligustica apiaries located at different altitudes in the Campania region (southern Italy): the Castelpoto Valley (BPv and BBv) and the Taburno Massif (BPm and BBm). Untargeted LC-MS/MS profiling enabled the identification of 50 phenolamides (mainly tri-caffeoyl, tri-coumaroyl, and caffeoyl-di-coumaroyl conjugates of spermidine and spermine) and 16 glycosylated flavonoids (primarily derivatives of isorhamnetin, quercetin, and kaempferol). Exploratory data analysis using Partial Least Squares showed that metabolites significantly vary according to both geographical site and matrix (BP vs BB). BPv had higher levels of di-p-coumaroyl spermidine and tetra-p-coumaroyl spermine isomers, while BPm and BBv were richer in tri-p-coumaroyl spermidine isomers. Pollen botanical origin was identified by Internal Transcribed Spacer 2 (ITS2) metabarcoding with Illumina Next-Generation Sequencing (NGS), which revealed significant differences in botanical composition between the collection sites and between BP and BB. BPv predominantly derived from Asteraceae and Fabaceae, while BBv samples were characterized by prevalent Fagaceae (mainly Castanea sativa) origin. BPm samples originated primarily from Boraginaceae and Fabaceae, while BBm samples were strongly dominated by Castanea sativa and Rosaceae. Overall, this study defines how geographic and botanical origin influence the content of bioactive compounds in BP and BB, highlighting the potential use of phenolic profiles as chemotaxonomic fingerprints and indicators of nutritional quality.