Toward stabilization of bioactive folate produced by indigenous lactic acid bacteria via BSA complexation

Folate deficiency remains a global health concern due to the instability and limited availability of its natural bioactive forms. Microbial production of reduced folate derivatives such as tetrahydrofolate (THF) and 5-methyltetrahydrofolate (5-MTHF) offers a sustainable means of providing the most bioavailable folate forms. This study screened lactic acid bacteria (LAB) from Indonesian kefir grains for extracellular THF and 5-MTHF biosynthesis and evaluated protein-based stabilization using bovine serum albumin (BSA) as a model carrier. Among 29 isolates, Lactiplantibacillus plantarum RAL25 demonstrated the highest 5-MTHF production (20.94 ± 8.36 µg/mL) in folate-free medium. The partially purified 5-MTHF fraction exhibited functional groups consistent with the authentic standard, and spectral evidence indicated its interaction with BSA. Molecular simulations further supported a stable protein-ligand association mediated by hydrogen bonding and hydrophobic forces. The combined investigation of natural 5-MTHF production by kefir-derived LAB and its stabilization through BSA complexation provides an integrated perspective that remains underexplored. These findings identify L. plantarum RAL25 as a promising natural 5-MTHF producer and establish a mechanistic basis for protein-assisted stabilization of reduced folates. Further validation through quantitative stability assays and bioavailability studies is warranted to advance this concept toward functional food applications.