Participants in a randomized controlled trial who followed a plant-heavy version of the Mediterranean diet saw a sharp rise in serum folate, a B vitamin tied to heart and metabolic health. The results came from the DIRECT PLUS trial, which tested whether adding green tea, walnuts, and a daily shake made from Wolffia globosa duckweed, known as Mankai, while cutting red meat could outperform a standard Mediterranean diet on key blood markers. The folate increase tracked with reductions in liver fat and shifts in gut bacteria, raising a pointed question: is the vitamin boost driven by the polyphenol-rich additions, by the drop in red meat, or by some combination that researchers have not yet pulled apart?
Why the green-MED folate spike demands a closer look
Folate and vitamin B12 sit at the center of one-carbon metabolism, the biochemical pathway that donates methyl groups to DNA. When that pathway works well, it helps regulate gene expression. When it falters, the consequences range from elevated homocysteine levels to disrupted epigenetic patterns linked to cardiovascular disease. The DIRECT PLUS trial, registered as NCT03020186, was designed to test whether a greener version of the Mediterranean diet could move these markers more than the traditional version.
The green-MED arm did exactly that. Serum folate climbed in participants who consumed the modified diet, and the rise correlated with measurable drops in intrahepatic fat, according to results published in the journal Gut. A separate analysis from the same trial connected the dietary changes to shifts in gut microbiome composition, suggesting the benefits extended beyond simple nutrient intake.
But the trial bundled several interventions together: polyphenol-rich green tea, walnuts, the Mankai shake, and reduced red meat. That bundling makes it difficult to isolate which element drove the folate increase. One plausible mechanism is that cutting red meat freed up methyl-group donors that would otherwise be consumed during the metabolism of animal protein. If that explanation holds, the polyphenols in green tea and Mankai may matter less for folate status than the dietary subtraction itself. Testing this would require a follow-up arm that swaps Mankai for an isocaloric, low-polyphenol plant protein while keeping meat intake the same. No such arm has been reported.
DIRECT PLUS trial findings and the Mankai factor
The DIRECT PLUS trial randomized participants into three groups: a healthy dietary guidelines arm, a traditional Mediterranean diet arm, and the green-MED arm. The green-MED group received the same caloric and macronutrient targets as the standard Mediterranean group but added daily green tea, a Mankai duckweed shake, and walnuts while further restricting red and processed meat. The polyphenol-enriched protocol and reduced meat intake together defined what made the diet “greener.”
Mankai itself has drawn independent scientific attention. A study in Nutrients confirmed that Wolffia globosa duckweed provides absorbable vitamin B12 in humans. That finding matters because plant-based diets typically lack reliable B12 sources, and B12 works alongside folate in the same methylation cycle. Researchers have traced the vitamin to a community of bacteria living inside the plant’s tissues, which produce cobamide compounds that Mankai accumulates.
Within DIRECT PLUS, investigators framed folate and B12 together as key drivers of the epigenetic effects observed in the green-MED group. A peer-reviewed analysis in Metabolism positioned these two vitamins as central to the diet’s ability to shift DNA methylation patterns. The implication is that the green-MED diet may influence disease risk not just through conventional nutrient pathways but through changes in how genes are read and expressed, especially in pathways related to lipid handling and inflammation.
The trial also reported that the green-MED group experienced greater reductions in visceral fat compared with both the standard Mediterranean and guideline-based groups. That result, combined with the folate and microbiome data, suggests a package effect rather than a single-nutrient story. Green tea catechins, walnut-derived unsaturated fats, Mankai’s B12 and protein, and the absence of red and processed meat each contribute something, but the relative weight of each input remains an open question.
Untangling nutrients from dietary patterns
One challenge in interpreting the DIRECT PLUS findings is that folate and B12 are both sensitive to overall dietary patterns. Leafy greens, legumes, and fortified grains all supply folate, while B12 typically comes from animal products or supplements. In the green-MED arm, participants likely increased their intake of folate-rich plants at the same time they added Mankai and reduced meat. That makes it difficult to attribute the serum folate rise to any one food, even though Mankai itself contains folate and B12.
The interaction between folate and B12 further complicates the picture. Adequate B12 is required to recycle folate into its active forms; if B12 is deficient, folate can become trapped in an unusable state, and homocysteine levels rise. The Nutrients data on Mankai’s B12 content support the idea that the duckweed shake could stabilize this cycle, allowing higher folate intake to translate into more effective methylation. However, DIRECT PLUS did not include a Mankai-free, meat-restricted arm with matched folate intake, so the independent contribution of the duckweed remains speculative.
Microbiome changes add another layer. Gut bacteria can synthesize certain B vitamins and modulate how much folate is absorbed or degraded in the intestine. The green-MED diet’s effect on microbial composition-linked in the Gut report to reductions in liver fat-could have altered local folate metabolism in ways not captured by simple intake measures. Yet without detailed metabolomic profiling and functional assays, those microbial contributions remain a black box.
Gaps in the green-MED evidence and what to watch next
Several limitations in the published record deserve attention. The exact serum folate concentration changes, measured in nanomoles per liter, appear in summary form across the trial’s papers but have not been released as participant-level data in publicly accessible tables. That makes independent reanalysis and subgroup exploration difficult, especially for people with baseline folate deficiency or genetic variants affecting folate transport.
Baseline B12 status and detailed dietary B12 intake for the Mankai arm are similarly underreported in the public materials, leaving a gap in understanding how much of the observed change reflects correcting subtle B12 insufficiency versus enhancing already adequate status. Without that context, it is hard to know whether the green-MED protocol mainly benefits those starting with low B-vitamin reserves or offers incremental advantages even for people with normal levels.
Another open question is durability. DIRECT PLUS followed participants for a defined intervention period, but data on long-term adherence to daily Mankai shakes and sustained meat restriction are scarce. If the folate and B12 advantages fade when people revert to more conventional eating patterns, the clinical relevance of the trial’s biochemical shifts could be limited. Longer follow-up studies would clarify whether the observed changes translate into fewer cardiovascular events, improved glycemic control, or reduced incidence of fatty liver disease.
Finally, the bundled design of the green-MED arm makes it inherently difficult to translate into everyday practice. Clinicians and patients may reasonably ask whether they need all components-green tea, walnuts, Mankai, and strict meat limits-to see meaningful benefits, or whether a simpler, more flexible pattern could achieve similar results. Factorial trials that vary these elements independently, or pragmatic studies that test streamlined versions of the diet in real-world settings, would help answer those questions.
What this means for nutrition science and practice
The DIRECT PLUS experience underscores both the promise and the complexity of food-based interventions. A plant-forward Mediterranean pattern enriched with specific polyphenols and an unusual aquatic plant appears capable of shifting folate and B12 status, reshaping the microbiome, and reducing liver and visceral fat. At the same time, the mechanisms behind those changes remain only partially mapped, and key data needed for rigorous independent evaluation are still missing.
For now, the safest takeaway is conceptual rather than prescriptive: combining diverse plant foods, limiting red and processed meat, and paying explicit attention to B-vitamin sufficiency-potentially including novel sources like Mankai-can nudge metabolic pathways in a favorable direction. As more granular data emerge and new trials disentangle individual components, clinicians and researchers will be better positioned to refine these patterns into targeted, evidence-based strategies for cardiometabolic and liver health.
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*This article was researched with the help of AI, with human editors creating the final content.
