Weissella cibaria strains: From selection of riboflavin-overproducing mutants to application for plant-based beverage fermentation.

Development of new plant-based functional beverages is currently increasing, and their biofortification by bacterial fermentation instead of chemical addition opens many possibilities. In this context, lactic acid bacteria (LAB) producing, in food matrices, essential micronutrients (i.e., vitamin B2 (riboflavin)), the immunomodulatory polysaccharide dextran, and other prebiotic and postbiotic compounds are valuable for the development of multifunctional fermented foods. Thus, with the aim of identifying and testing new LAB strains, three spontaneous riboflavin-overproducing mutants were selected by roseoflavin treatment of the dextran- and mannitol-producing Weissella cibaria V30 strain. The mutants showed various levels of increased riboflavin synthesis. Riboflavin biosynthesis in LAB is performed by the enzymes encoded by the rib operon, whose expression is regulated at an FMN-responsive riboswitch and the three mutants carry single-nucleotide substitutions affecting the folding of the riboswitch FMN-binding aptamer. V30 C29A displayed the highest production of riboflavin (up to 5.8 mg/L), more than 20-fold higher than that of V30, with efficient extracellular vitamin secretion (∼90%) while retaining robust dextran production (6.2 g/L). Fermentation of three commercial drinks based on oat, soy or rice with V30 C29A resulted in a matrix-dependent accumulation of vitamin B2 and dextran, with the highest yields (5.5 mg/L and 3.3 g/L, respectively) in the oat beverage. Metabolic profiling revealed the production of additional functional metabolites, including the prebiotic oligosaccharides panose and isomaltose as well as mannitol. These findings identify W. cibaria V30 C29A as a promising candidate for in situ biofortification of non-dairy beverages under the conditions tested, enhancing their nutritional and functional value.