Vitamin B12 biofortification of soymilk through optimized fermentation with extracellular B12 producing Lactobacillus isolates of human fecal origin

Abstract

The present study was designed to bio-fortify the soymilk (<i>per se</i> a B12-free plant food matrix). The PCR-based screening characterized the human fecal samples (4 out of 15 tested) and correspondingly identified novel lactobacilli isolates (n = 4) for their B12 production potential and rest (n = 62) as negative for this attribute. Further, 3 out of the 4 selected strains showed ability for extracellular vitamin production. The most prolific strain, <i>Lactobacillus reuteri</i> F2, secreted B12 (132.2 ± 1.9 μg/L) in cobalamin-free-medium with the highest ratio ever reported (0.97:1.00; extra-: intra-cellular). In next stage, the soymilk was biofortified <i>in situ</i> with B12 during un-optimized (2.8 ± 0.3 μg/L) and optimized (156.2 ± 3.6 μg/L) fermentations with a ∼54-fold increase at Artificial Neuro Fuzzy Inference System based R value of >0.99. The added-nutrients, temperature and initial-pH were observed to be the most important fermentation variables for maximal B12 biofortification. We report <i>Lactobacillus rhamnosus</i> F5 as the first B12 producing (101.7 ± 3.4 μg/L) strain from this species. The cyanocobalamin was extracted, purified and separated on UFLC as nutritionally-relevant B12. Besides, the vitamin was bioavailable in an auxotrophic-mutant. The lactobacilli fermentation is suggested, therefore, as an effective approach for B12 biofortification of soymilk.