Untargeted metabolomic analysis was performed to identify the principal aglycone generated by the engineered Escherichia coli Nissle 1917 strain expressing β-galactosidase (EcN-bgal). Culture supernatants from EcN-bgal and the vector control strain were collected and subjected to LC–MS-based metabolomic profiling. Comparative analysis revealed substantial alterations in the metabolite composition of the EcN-bgal supernatant, enabling the identification of β-galactosidase-dependent metabolites and the characterization of the major aglycone produced by the engineered strain.