Spontaneous riboflavin-overproducing Limosilactobacillus reuteri for biofortification of fermented foods. undefined

Lactic acid bacteria producing riboflavin (vitamin B2) represent a promising and cost-effective strategy for food biofortification, but production levels are typically insufficient to support daily human requirements. In this study, we describe the novel human isolate Limosilactobacillus reuteri AMBV339 as a strong food biofortification candidate. This strain shows a high natural riboflavin overproduction of 18.36 µg/ml, biomass production up to 6x1010 colony-forming units/ml (in the typical range of model lactobacilli), and pH-lowering capacities to a pH as low as 4.03 in common plant-based (coconut, soy and oat) and milk beverages when cultured up to 72 h at 37°C. These properties were especially pronounced in coconut and butter milk fermentations and were sustained in co-culture with the model starter Streptococcus thermophilus. Furthermore, L. reuteri AMBV339 grown in laboratory media or a coconut beverage survived in gastric juice (almost 100% survival at pH 3 and 4) and in a simulated gastrointestinal dialysis model with colon phase (GIDM-model) inoculated with fecal material from a healthy volunteer. Passive transport of L. reuteri AMBV339-produced riboflavin occurred in the small intestinal and colonic colon stage of the GIDM system, and active transport via intestinal epithelial Caco-2 monolayers was also demonstrated. L. reuteri AMBV339 did not cause fecal microbiome perturbations in the GIDM system and inhibited enteric bacterial pathogens in vitro. Taken together, our data suggests that L. reuteri AMBV339 represents an exceptional candidate to provide riboflavin fortification of cow milk- and plant-based foods and has a high application potential in the human gastrointestinal tract.