Simple analytical approach to determine the microbiological profile of bee pollen by MALDI-TOF

Bee pollen is a natural food supplement collected by honeybees, has significant nutritional value and therapeutic qualities, including antioxidant, antibacterial, and anticarcinogenic properties. Due to its hygroscopic nature, it easily promotes microorganisms’ growth; nevertheless, there is no specific legislation for bee pollen in the European Union. Microbiological contamination stands out as a crucial factor in determining its quality. To address this, the study focused on isolating bacteria and fungi colonies from bee pollen using various culture media, followed by a protein extraction based on a lysis process. Identification was achieved through the Biotyper-MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization coupled to Time-of-Flight detector) system. The greenness and blueness of the methods was assessed with different tools that classified it as environmentally friendly and practically. Thirty-two samples were analyzed using proposed sample treatment approaches. Data were processed using chemometric techniques based on linear models. The results revealed the presence of both beneficial and potentially pathogenic species, including key representatives such as Bacillus licheniformis , Micrococcus luteus , and Aspergillus fumigatus. These findings highlight the importance of microbiota characterization in bee pollen and demonstrate that MALDI-TOF is a rapid and effective tool for profiling culturable microbial communities in this matrix. • MALDI-TOF was used to identify both bacterial and fungal isolates in bee pollen. • Fungal protein extraction was optimized by using ceramic homogenizer. • Greenness was evaluated with AGREE, AGREEprep, MoGAPI, and BAGI tools. • PCA and HCA revealed differences between commercial and experimental samples. • Microbiological profiling demonstrated potential for differentiating samples origin.