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

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.

Polymerase chain reaction for molecular detection of the genes involved in the production of riboflavin in lactic acid bacteria.

Some lactic acid bacteria (LAB) strains have the ability to synthesize riboflavin, a trait linked to the presence of ribG, ribB, ribA and ribH genes in the rib operon. Multiple sequence alignments of these genes showed that these sequences are not identical in different LAB species, so primers designed to detect these genes in one species do not always work with others. Therefore, we designed degenerate primers based on sequences from Lactococcus lactis MG1363, Levilactobacillus brevis ATCC 367 and Limosilactobacillus fermentum IFO3956, and established optimal PCR conditions for the detection of rib genes in different LAB species. Simultaneously, we selected riboflavin-producing LAB strains from our bacterial collection belonging to the species L. brevis, L. fermentum, L. lactis, Leuconostoc mesenteroides and Lactiplantibacillus plantarum, and we were able to detect ribG, ribB, ribA and ribH genes in these strains by PCR using the designed primers. Thus, the development of degenerate primers and optimal PCR conditions for the detection of ribG, ribB, ribA and ribH genes in LAB allowed the detection and the selection of potential riboflavin-producing strains of different species, which could be good candidates for the development of riboflavin-enriched functional foods.