Characterization of riboflavin-overproducing Bifidobacterium longum subsp. infantis strains selected by roseoflavin treatment

Diet is the primary source of riboflavin (B₂) for humans. It can also be produced by lactic acid bacteria ingested with foods and by gut microbial commensals, including some bifidobacteria. Herein an in silico analysis of potential regulatory mechanisms affecting ribD transcription and translation in Bifidobacterium longum subsp. infantis is presented. Riboflavin-overproducing strains were selected by treatment with roseoflavin of B. longum susbp. infantis CECT4551^T and its spontaneous derivative IPLA60011. Whole genomes of both parental strains and the sequencing of the rib clusters of the riboflavin-overproducing ones were conducted. Punctual mutations affecting different stem-loops in the aptamer region of the FMN-riboswitch involved in the regulation of the rib expression were detected. Riboflavin overproduction of the derivative strains was confirmed through HPLC quantification in RAMc and MRSc cultures, ranging from 64.9 to 441.2 μg/L. These levels correlated to predicted secondary folding and stability of the aptamer region and/or expression platform of the rib FMN riboswitch. Safety and technological properties of the riboflavin-overproducing derivatives, in terms of antibiotic resistance profile and carbohydrate utilization capabilities, were not altered following roseoflavin exposure, thus confirming the potential aptitude of the riboflavin-overproducing derivatives to produce biofortified foods such as those formulated on dairy matrixes.