Droplet microfluidics-based isolation, adaptation, and screening of riboflavin-producing lactic acid bacteria for fermenting plant-based dairy alternatives

The development of novel starter cultures is essential for enhancing the nutritional value of plant-based dairy alternatives. In this study, we developed an innovative screening method to isolate lactic acid bacteria capable of in situ riboflavin production in soy-based substrates. Our approach integrates high throughput (HTP) single-cell cultivation, adaptation, and fluorescence-activated droplet sorting system. This method bypasses initial microbe isolation, involving all microorganisms from a sample for screening. By combining this workflow with our newly developed transparent soy-based medium, we efficiently identified LAB strains from bee gut that are suitable for soy-based fermentation. Among these strains, Lactococcus lactis NFICC2835 demonstrated the best performance during the screening process. By characterizing NFICC2835 in commercial soy-based milks, it showed consistent riboflavin production with up to 1.23 mg/L revealed by HPLC. Genomic analysis of NFICC2835 revealed 13 missense mutations around the rib operon and the purH gene, and 3 mutations in regions critical for regulating riboflavin biosynthesis, including the FMN riboswitch. By showcasing the genetic characteristics of the strains obtained from the HTP droplet screening, we not only advance our understanding of riboflavin overproduction but also underscore the potential of our screening strategy over conventional approaches in accelerating strain discovery and functional food development. • Developed a soy-based culture medium compatible with microfluidic screening. • Demonstrated a screening method that skips strain isolation from the beginning. • Demonstrated a method for microbial adaptation and screening at a community level. • Demonstrated a high-throughput method for screening LAB directly from gut samples. • Identified mutations linked to enhanced riboflavin biosynthesis in LAB.