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D’Aimmo et al. Microbiome Res Rep 2024;3:11 https://dx.doi.org/10.20517/mrr.2023.59 Page 7 of 22

[43]
has no noticeable impact on folate levels . From a public health perspective, providing practical advice on
cooking techniques may be a useful strategy to improve folate intake.

In addition to chemical breakdown, folates can undergo structural changes through molecular
interconversion from one form to another, potentially affecting their bioavailability [43,44] ; according to a
study by O’Broin et al. , folate stability depends mainly on the one-carbon unit in the molecular structure,
[45]
where 5-HCO-THF is more stable than 5-methyl-THF, followed by 10-HCO-THF and THF. However, the
authors quantified folates using a microbiological assay that detects all biologically active forms without
qualitative information. Therefore, it cannot be ruled out that the higher stability of 10-HCO-THF over
THF was due to the conversion of 10-HCO-THF to more stable 5-HCO-THF and/or 10-HCO-folic acid.

Folic acid has greater stability in comparison with the reduced folate forms. Although some folate oxidation
resulting in folic acid formation may occur during food storage or cooking, folic acid is virtually unknown
[46]
in nature . This is because THF or H -folate would rarely lose their hydrogen and form folic acid while
2
[47]
remaining uncleaved .
ANALYSIS OF FOLATE
The methods of analyzing folates have been thoroughly reviewed elsewhere [48,49] , and we will only give a brief
overview to emphasize some critical points [Supplementary Material]. The main steps when assessing
folates in biomaterials such as microbial biomass, foods, or blood serum are: (i) extraction from the
biological matrix; (ii) deconjugation of the polyglutamate tail resulting in mono-glutamate-folates; and (iii)
detection of the resulting released folates. Depending on the method used, it will yield either a
quantification of total folates, i.e., all bioactive derivatives counted as one, or quantitative information on
each folate derivative. If total bioactive folate content is sufficient, this commonly means using a
microbiological assay (MA) . The basis for the MA is to select a microorganism that requires an external
[49]
supply of folate and is unable to synthesize the folate by itself. Assuming all other necessary components are
available in the medium, the growth will depend on the quantity of folate present in the added sample. By
comparing the growth responses between unknown samples and folate standards, folate can be quantified.
The MA is very sensitive (sub-nanogram levels) and does not require advanced analytical instruments.
However, it is time-consuming and strictly quantitative and will therefore not give information on the
relative concentration of different folate forms. Choosing the right species and strain of microorganisms for
the MA assay is critical since it depends on equal response on growth for the different folate derivatives.
Lacticaseibacillus rhamnosus ATCC 7469 has been shown to yield equal responses to different folate forms
and has hence become standard.

If detailed information is needed, high-performance liquid chromatographic (HPLC) tends to be
preferred . By using suitable columns and methods, HPLC enables the separation and detection of
[50]
individual different folate forms (typically detected by UV absorbance, fluorescence, or mass spectrometry)
but requires expensive equipment and expertise [Supplementary Material].

HUMAN FOLATE GENES AND EVOLUTION
Various environmental factors have influenced the evolution and expansion of modern humans, including
the climate and diet.

During the Paleolithic era, hunting and gathering were the sole methods of subsistence, and human dietary
adaptations were heavily impacted by food availability. Notably, groups inhabiting ice-age climates
experienced recurring food shortages during winter and spring, resulting in a decreased intake of plant