Wolffia globosa-Mankai Plant-Based Protein Contains Bioactive Vitamin B12 and Is Well Absorbed in Humans

Abstract

Background: Rare plants that contain corrinoid compounds mostly comprise cobalamin analogues, which may compete with cobalamin (vitamin B₁₂ (B₁₂)) metabolism. We examined the presence of B₁₂ in a cultivated strain of an aquatic plant: Wolffia globosa (Mankai), and predicted functional pathways using gut-bioreactor, and the effects of long-term Mankai consumption as a partial meat substitute, on serum B₁₂ concentrations.

Methods: We used microbiological assay, liquid-chromatography/electrospray-ionization-tandem-mass-spectrometry (LC-MS/MS), and anoxic bioreactors for the B₁₂ experiments. We explored the effect of a green Mediterranean/low-meat diet, containing 100 g of frozen Mankai shake/day, on serum B₁₂ levels during the 18-month DIRECT-PLUS (ID:NCT03020186) weight-loss trial, compared with control and Mediterranean diet groups.

Results: The B₁₂ content of Mankai was consistent at different seasons (p = 0.76). Several cobalamin congeners (Hydroxocobalamin(OH-B₁₂); 5-deoxyadenosylcobalamin(Ado-B₁₂); methylcobalamin(Me-B₁₂); cyanocobalamin(CN-B₁₂)) were identified in Mankai extracts, whereas no pseudo B₁₂ was detected. A higher abundance of 16S-rRNA gene amplicon sequences associated with a genome containing a KEGG ortholog involved in microbial B₁₂ metabolism were observed, compared with control bioreactors that lacked Mankai. Following the DIRECT-PLUS intervention (n = 294 participants; retention-rate = 89%; baseline B₁₂ = 420.5 ± 187.8 pg/mL), serum B₁₂ increased by 5.2% in control, 9.9% in Mediterranean, and 15.4% in Mankai-containing green Mediterranean/low-meat diets (p = 0.025 between extreme groups).

Conclusions: Mankai plant contains bioactive B₁₂ compounds and could serve as a B₁₂ plant-based food source.

Keywords: Wolffia globosa; flexitarians; plant-based nutrition; sustainability; vitamin B12; weight loss.

Figure 1

Stability of vitamin B₁₂ levels in Mankai™ along the year. “Autumn” refers to water temperatures of 22–24.5 °C and 10:20–10:50 h of light. “Winter” refers to water temperatures of 17–20 °C and 10–10:20 h of light. “Spring” refers to water temperatures of 21–24 °C and 11:30–13:30 h of light. “Summer” refers to water temperatures of 25–29 °C and 13:50–14:15 h of light. For each season, the weekly average water temperatures and daily light hours relate to the sampling date.

Figure 2

Liquid chromatography/electrospray ionization tandem mass spectrometry (LC-MS/MS) chromatograms of CN-B₁₂. (A) Retention time for CN-B₁₂ standard (arrow). (B) Retention time for CN-B₁₂ extracted from Mankai sample (arrow). ES, electrospray; MRM, multiple reaction monitoring; TIC, total ion current.

Figure 3

A comparison of chromatograms of TIC for authentic CN-B₁₂ and pseudo CN-B₁₂ in Mankai and spirulina samples. (A–C): Active CN-B₁₂; (B–D) Pseudo CN-B₁₂ in Mankai™ (A,B) and spirulina (C,D) samples. In panel B, a peak at 2.12 min does not represent pseudo CN-B₁₂ because pseudo CN-B₁₂ should appear before the peak of CN-B₁₂ [27,28] as is observed with a peak from a spirulina sample (at 2.09 min, panel D) and is present not just in one but in all 4 MRM transitions at measurable levels (Figure S4). ES, electrospray; MRM, multiple reaction monitoring; TIC, total ion current.

Figure 4

The 18-month change in serum vitamin B₁₂ across intervention groups. * Indicates within-group change (baseline vs. T18) at the 0.05 level. Data presented as means and SEM. HDG, healthy dietary guidelines; MED, Mediterranean.

Figure 5

Red meat consumption change at the end of the intervention (tertiles) vs. 18-month serum folate change (tertiles) vs. 18-month change in vitamin B_12. * indicated within-group significance (baseline vs. T18) at the 0.05 level. Data presented as means and SEM.