2.2. Bee Pollen Samples and Palynological Analysis

Five bee pollen samples (Apis Mellifera), named as P01, P02, P03, P04, and P05, were harvested in 2016 and 2017 from two different geographical areas of Tuscany (Italy), in particular Garfagnana (LU) and Caniparola-Fosdinovo (MS), by two different Tuscan farms, specifically “Apicoltura Biologica Aldo Metalori” (Massa Macinaia, LU, Italy) and “Azienda Apistica Guidarelli Andrea” (Fivizzano, Massa Carrara, MS, Italy). Among these five samples, composed by a mix of colored grains, bee pollens were divided based on the grain color and only those presenting a distinct homogeneous color were analyzed to confirm their monofloral character. The list of bee pollen samples, further divided for their botanical origin, is reported in Table 1. All samples underwent a light heat treatment (T_max = 38 °C) to reduce the free water content before being stored at −20 °C in the dark until further analysis.

List of the bee pollen samples investigated in this work. The sample label reflects the sample of origin (P0X) followed by one of two letters to indicate their botanical origin. The botanical origin is expressed in %, the geographical origin, the color, and year and month of harvesting are also reported. Each pollen load owned a homogeneous and monospecific pollen content.

The botanical origin of the bee pollen samples was confirmed by the melissopalynology analysis by means of an optical microscope. Each single pollen load was washed with distilled water and fixed with glycerin jelly. Bee pollen grains identification was performed by optical microscope (Leica DME, Buccinasco (MI), Italy) with total magnification (400X and 1000X). A reference collection of Tor Vergata University and different pollen morphology guides were used for the recognition of pollen [31].