| Summary: | A variety of bioactive compounds, constituents of edible mushrooms, in particular β-glucans, i.e., a group of β-<span style="font-variant: small-caps;">d</span>-glucose polysaccharides abundant in the fungal cell walls, have been linked to immunomodulating, anticancer and prebiotic activities. The aim of the study was the investigation of the genoprotective effects of edible mushrooms produced by <i>Pleurotus eryngii</i>, <i>Pleurotus ostreatus</i> and <i>Cyclocybe cylindracea</i> (Basidiomycota). Mushrooms from selected strains of the species mentioned above were fermented in vitro using faecal inocula from healthy volunteers. The cytotoxic and anti-genotoxic properties of the fermentation supernatants (FSs) were investigated in Caco-2 human colon adenocarcinoma cells. The FSs were cytotoxic in a dose-dependent manner. Non-cytotoxic concentrations were used for the genotoxicity studies, which revealed that mushrooms’ FSs have the ability to protect Caco-2 cells against tert-butyl hydroperoxide (<i>t</i>-BOOH), a known genotoxic agent. Their global metabolic profiling was assessed by <sup>1</sup>H-NMR spectroscopy. A total of 37 metabolites were identified with the use of two-dimensional (2D) homo- and hetero-nuclear NMR experiments. Multivariate data analysis monitored the metabolic variability of gut microbiota and probed to biomarkers potentially associated with the health-promoting effects of edible mushrooms.
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