Bee derived Aspergillus oryzae as a novel reservoir for selective anticancer metabolites with integrated bioprocess optimization and multi cell line evaluation

Cancer is a major global health challenge, driving the need for new therapeutic agents. This study explored the anticancer potential of microorganisms isolated from healthy bee workers collected from 35 sites in three Egyptian governorates to identify novel bioactive metabolites. A total of 113 microbial isolates (35 fungal and 78 bacterial) were obtained and screened. Thirty-two selected isolates were fermented, and their cytotoxicity against MCF-7 breast cancer cells was assessed using the MTT assay. Eight isolates showed strong anticancer activity, reducing cell viability below 30%. Among them, isolate Tm2 demonstrated the highest selectivity and potency across multiple cancer cell lines (Caco-2, MCF-7, HepG-2, A549), with IC50 values between 30.63 and 113.39 µg/ml, and minimal toxicity toward normal cells (IC50 > 976 µg/ml; selectivity index = 18.21). Morphological and molecular analyses identified Tm2 as Aspergillus oryzae. Optimization of fermentation parameters via Plackett-Burman and Box-Behnken designs maximized extract yield to 1.86 g/100 ml under specific conditions (yeast extract 1.5 g/L, pH 7.6, 25 °C). Chemical profiling using GC-MS and UPLC-MS/MS detected 65 bioactive compounds, mainly fatty acids such as oleic, hexadecanoic, and linoleic acids. Live-dead staining showed dose-dependent cytotoxic effects, while flow cytometry revealed induction of apoptosis through multiple pathways in cancer cells treated with the extract. Aspergillus oryzae isolated from bee microbiota exhibits potent and selective anticancer activity mediated by a complex mixture of bioactive fatty acids. These results highlight its potential as a novel source for developing effective anticancer therapies with low toxicity to normal cells.