Riboflavin bio-enrichment of soy beverage by selected roseoflavin-resistant and engineered lactic acid bacteria

Abstract

Some lactic acid bacteria (LAB) have the ability to synthesize riboflavin, a trait linked to the presence of ribG, ribB, ribA and ribH genes located in the rib operon. Previous screening of riboflavin producers identified several LAB strains belonging to different species with this ability, but none of them surpassed 0.25 mg/L production of the vitamin. In this study, we explored two strategies to obtain riboflavin-overproducing strains: by roseoflavin selection of mutants, and by the transformation of selected strains with plasmids pNZ:TuR.rib or pNZ:TuB.rib containing the genes ribG, ribB, ribA and ribH from Lactococcus cremoris MG1363. The resulting riboflavin-overproducing strains were able to produce yields between 0.5 and 6 mg/L in culture media and several of them were selected for the fermentation of soy beverages. Riboflavin in bio-enriched soy beverages was evaluated by direct fluorescence measurement and high-performance liquid chromatography-fluorescence analysis. Soy beverages fermented with the recombinant strains Lactococcus cremoris ESI 277 pNZ:TuB.rib and Lactococcus lactis INIA 12 pNZ:TuR.rib showed the highest riboflavin yields (>5 mg/L) after 24 h fermentation. On the other hand, roseoflavin-resistant mutant Limosilactobacillus fermentum INIA P143R2 was able to enrich fermented soy beverages with 1.5 mg/L riboflavin. Riboflavin-overproducing LAB strains constitute a good option for riboflavin enrichment of soy beverages by fermentation and the commercialization of such beverages could be very useful to prevent riboflavin deficiency.