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The fruit-origin strain Fructobacillus tropaeoli CRL 2034 can biotransform selenium into seleno-nanoparticles and selenocysteine. The proteomic analysis of F. tropaeoli CRL 2034 exposed to 5 and 100 ppm of Se showed a dose-dependent response since 19 and 77 proteins were deregulated, respectively. In the presence of 5 ppm of Se, the deregulated proteins mainly belonged to the categories of energy production and conversion or had unknown functions, while when cells were grown with 100 ppm of Se, most of the proteins were grouped into amino acid transport and metabolism, nucleotide transport and metabolism, or into unknown functions. However, under both Se conditions, glutathione reductases were overexpressed (1.8–3.1-fold), while mannitol 2-dehydrogenase was downregulated (0.54–0.19-fold), both enzymes related to oxidative stress functions. Mannitol 2-dehydrogenase was the only enzyme found that contained SeCys, and its activity was 1.27-fold increased after 5 ppm of Se exposure. Our results suggest that F. tropaeoli CRL 2034 counteracts Se stress by overexpressing proteins related to oxidative stress resistance and changing the membrane hydrophobicity, which may improve its survival under (food) storage and positively influence its adhesion to intestinal cells. Selenized cells of F. tropaeoli CRL 2034 could be used for producing Se-enriched fermented foods. Graphical Abstract: [Figure not available: see fulltext.] Key points: • Selenized cells of F. tropaeoli showed enhanced resistance to oxidative stress. • SeCys was found in the Fructobacillus mannitol 2-dehydrogenase polypeptide chain. • F. tropaeoli mannitol 2-dehydrogenase activity was highest when exposed to selenium., Ministerio de Ciencia y Tecnología (Argentina), Ministerio de Ciencia e Innovación (España), Comunidad de Madrid, European Commission, Depto. de Química Analítica, Fac. de Ciencias Químicas, TRUE, pub