Gorse (Ulex europeaus) wastes with 5,6-dimethyl benzimidazole supplementation can support growth of vitamin B12 producing commensal gut microbes

Abstract

Many commensal gut microbes are recognized for their potential to synthesize vitamin B12, offering a promising avenue to address deficiencies through probiotic supplementation. While bioinformatics tools aid in predicting B12 biosynthetic potential, empirical validation remains crucial to confirm production, identify cobalamin vitamers, and establish biosynthetic yields. This study investigates vitamin B12 production in three human colonic bacterial species: Anaerobutyricum hallii DSM 3353, Roseburia faecis DSM 16840, and Anaerostipes caccae DSM 14662, along with Propionibacterium freudenreichii DSM 4902 as a positive control. These strains were selected for their potential use as probiotics, based on speculated B12 production from prior bioinformatic analyses. Cultures were grown in M2GSC, chemically defined media (CDM), and Gorse extract medium (GEM). The composition of GEM was similar to CDM, except that the carbon and nitrogen sources were replaced with the protein-depleted liquid waste obtained after subjecting Gorse to a leaf protein extraction process. B12 yields were quantified using liquid chromatography with tandem mass spectrometry. The results suggested that the three butyrate-producing strains could indeed produce B12, although the yields were notably low and were detected only in the cell lysates. Furthermore, B12 production was higher in GEM compared to M2GSC medium. The positive control, P. freudenreichii DSM 4902 produced B12 at concentrations ranging from 7 ng mL-1 to 12 ng mL-1. Univariate-scaled Principal Component Analysis (PCA) of data from previous publications investigating B12 production in P. freudenreichii revealed that B12 yields diminished when the carbon source concentration was ≤30 g L-1. In conclusion, the protein-depleted wastes from the leaf protein extraction process from Gorse can be valorised as a viable substrate for culturing B12-producing colonic gut microbes. Furthermore, this is the first report attesting to the ability of A. hallii, R. faecis, and A. caccae to produce B12. However, these microbes seem unsuitable for industrial applications owing to low B12 yields.

Fig 1. Structure of cobalamin vitamers.

Variable side-groups (S_v) form coordinate bonds with the central cobalt ion. The ligands at location L_l determine if the cobalamin vitamer is biologically active in humans with 5,6-dimethybenzimidazole (DMB) or inactive with adenine (marked in red). The cyano-group (marked in blue) is an example of semi-synthetic cobalamin derivates. B₁₂ vitamers can be found with combinations of S_v and L_l ligands.

Fig 2. Microbial growth profile.

Growth profile of A. caccae DSM 14662, A. hallii DSM 3353, P. freudenreichii DSM 4902, and R. faecis DSM 16840 in chemically defined medium (CDM), gorse extract medium (GEM) and M2GSC.

Fig 3. Dimension reduction of experimental conditions to elucidate their influence on B₁₂ yields.

Previously reported experimental conditions were clustered using k-means analysis. The clustering scheme was overlaid on a univariate-scale PCA model that was constructed using previously reported parameters for the growth of Propionibacterium freudenreichii, namely, 5,6-dimethylbenzimidazole (DMB, mg L⁻¹), cobalt (mg L⁻¹), total carbohydrates (Carbon, g L⁻¹), and incubation time (h). The point marked ‘E’ refers to the intrapolated position of the experimental conditions described herein and correspondingly, the expected B₁₂ yields, which appears to associate to cluster 2. Ellipses represent 95% confidence limits of each cluster. Data and codes used for dimension reduction analyses is provided in S2 File.