Nowadays, natural products have gained increasing attention from consumers due to their proven nutritional value and biological activity. Honeybees produce several products that have potential nutritional value and health benefits (Giampieri et al., 2022). In addition to the well-known honey, other bee products like bee pollen, propolis, bee bread, and royal jelly are also gaining attention. Bee pollen consists of pollen grains attached to the hind legs of bees during foraging, as well as nectar and salivary substances from bees (Friedle et al., 2021). In addition to being the main food for bees, bee pollen is also considered a good dietary supplement for humans because it is rich in nutrients - proteins, fatty acids, carbohydrates, vitamins, phytosterols, polyphenolics, and minerals (Algethami et al., 2022; Laaroussi et al., 2023). Along with its prominent nutritional benefits, bee pollen also has potential therapeutic properties of antioxidant, anti-inflammatory, antiallergenic, antimicrobial, anti-atherosclerotic, and anticancer (Algethami et al., 2022; Denisow and Denisow-Pietrzyk, 2016). Research has shown that bee pollen has preventive effects on metabolic disorders such as obesity, diabetes, atherosclerosis, and cardiac damage (Cheng et al., 2019; Khalifa et al., 2021; Li et al., 2021; Rzepecka-Stojko et al., 2017). Considering its well-known nutritional and medicinal values, bee pollen has recently been used as a food supplement and cosmetic in the food and cosmetic fields, respectively (Algethami et al., 2022; Conte et al., 2018).

The safety of bee pollen has been a hot topic of research in recent years. Given its rich nutrient content, a range of microorganisms can grow and proliferate in bee pollen. Fungi can grow in bee pollen if it is collected, dried, processed and stored inappropriately, resulting in mildew and mycotoxin production (Friedle et al., 2021; Kostić et al., 2019). Observation of macroscopic and microscopic characteristics of isolates is the classical approach for fungal taxonomic identification in bee pollen (Deveza et al., 2015; González et al., 2005; Kačániová et al., 2011; Nardoni et al., 2016). Previous research showed that Cladosporium spp. and Penicillium chrysogenum were the most common fungi isolated from 40 specimens of bee pollen in Central Italy (Nardoni et al., 2016). Aspergillus, Cladosporium, and Penicillium were the dominant genera isolated from 27 commercial bee pollen samples in Brazil (Deveza et al., 2015). However, a non-negligible drawback of the cultivation-dependent method is that many microorganisms are not culturable (Daniel, 2004). The emergence of DNA metabarcoding combines the advantages of high-throughput sequencing and DNA taxonomy, allowing for the simultaneous supervision of multiple species from a single sample (Taberlet et al., 2012; Galimberti et al., 2014). The emergence of DNA metabarcoding provides a new way to fully understand the microbes in bee pollen (De Jesus Inacio et al., 2021; Friedle et al., 2021).

In recent years, there have been frequent reports of mycotoxin contamination in bee pollen, which poses a latent threat to public health. Aflatoxins (AFs), deoxynivalenol (DON), zearalenone (ZON), ochratoxins (OTs), and T-2 toxin have previously been observed in rape bee pollen, poppy bee pollen, and sunflower bee pollen samples (Kačániová et al., 2011). In Spain, two out of 15 commercial bee pollen samples collected were positive for nivalenol (NIV) and neosolaniol (NEO) (Rodríguez-Carrasco et al., 2013). Keskin and Eyupoglu (2023) analyzed HT-2 toxin, T-2 toxin, Ochratoxin A (OTA), and DON in 28 batches of fresh bee pollen samples in Turkey, with the detection rate of the four mycotoxins not <17 %. Among the reported mycotoxins, AFs are the most toxic, with hepatotoxicity, carcinogenicity, immunotoxicity, and nephrotoxicity (Afshar et al., 2020; Moore et al., 2018). Meanwhile, high detection rates of AFs in bee pollen samples from many countries have been frequently reported (Carrera et al., 2023; Petrovic et al., 2014). Therefore, investigation of AFs contamination in bee pollen is crucial to guarantee its safe use. High-performance liquid chromatography-fluorescence detection (HPLC-FLD) equipped with a post-column photochemical reactor is widely recognized as one of the most commonly employed methods to detect AFs in various food and herbal medicine samples (Shuib et al., 2017; Simão et al., 2016; Wang et al., 2023), providing a simple, feasible, and accurate way for the monitoring of AFs in bee pollen.

As is well known, the color of pollen grains varies greatly, reflecting the species diversity of the plants visited by bees (Deveza et al., 2015). Types of bee pollen are generally distinguished according to the botanical source of the pollen, such as rose bee pollen, multiflora bee pollen, buckwheat bee pollen, and so on. At present, information on fungal diversity and AFs contamination in different types of bee pollen in China is almost vacant. In this study, we aim to reveal the mycobiota in five types of commercial bee pollen samples from eight provinces and one autonomous region of China by targeting the ITS2 region on the Illumina MiSeq platform and to simultaneously investigate the residual levels of total AFs (AFB1, AFB2, AFG1, and AFG2) in each sample using HPLC-FLD by pretreatment with an immunoaffinity column (IAC). This study offers valuable insights into the simultaneous monitoring of fungal communities and mycotoxins in bee pollen samples. The findings have significant implications for ensuring the safe utilization of bee pollen.