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Page 10 of 22 D’Aimmo et al. Microbiome Res Rep 2024;3:11 https://dx.doi.org/10.20517/mrr.2023.59

Nonetheless, establishing a clear connection between the existence of folate biosynthesis machinery and its
actual in vivo activity level presents a challenging and currently elusive task. From our perspective, there is a
substantial need for further research to determine the extent to which the gut microbiota contributes to
human folate intake.

THE PRODUCTION OF FOLATE IN BIFIDOBACTERIA
The genus Bifidobacterium is considered one the most important bacterial group of the gut microbiota,
exerting various health-promoting effects on its host: bifidobacteria supports the development of the host
immune system, promote intestinal barrier integrity, and provide protection against pathogen growth [72,73] .
The Bifidobacterium genus includes several species capable of de novo folate biosynthesis [58,65,74-76] .

Folate content in a particular bacterium is not a static property unless conditions are steady, such as in a
chemostat, which is never the case in real-life scenarios. Moreover, another significant factor is that the
folate content can change significantly based on various factors, such as the type and degree of cellular
activity, population state, growth rate, pH, temperature, and medium composition [77-79] . For example, folate-
producing LAB strains exhibited a temperature-dependent pattern. At 42 °C, they achieved maximum folate
production in just 6 h, whereas at 37 °C, it took 8 h to reach the same level. This highlights the crucial role
of temperature control in optimizing folate production in LAB strains . Controlled pH conditions favored
[80]
the synthesis of folate in Streptococcus macedonicus CRL415: In fact, the total amount of folate produced
raised ca. 2-fold when the pH was increased from 5.0 to 6.0 with respect to free-pH . Therefore, the folate
[81]
content of a bacterium is highly variable and subject to change. Another important factor contributing to
the diversity is the variation in folate analysis methods used in different studies. Finally, factors contributing
to biodiversity are natural and intrinsic gene variation. This is supported by research on numerous strains
under identical conditions in a single laboratory [74,82] .

The predominant phenotype observed in certain species is linked to functional folate-synthesizing
machinery. Among these species, some have consistently demonstrated a high specific folate content,
measured as the weight of folate per dry weight of bacterial biomass. Notably, B. adolescentis, B. bifidum,
B. catenulatum, and B. pseudocatenulatum are among the studied species with a dominant autotrophic
ability for folate synthesis.

In B. catenulatum ATCC 27539, the highest value of folate production previously found was 93 µg per g DM
(the highest in yeast is approximately 200 µg per g DM). The substantial diversity observed in yeasts and
bifidobacteria can be attributed to technical and biological factors. As a point of comparison, broccoli,
renowned for its folate content, typically contains about 70-80 µg of folate per 100 g of fresh weight.
Considering its water content of about 87%, this translates to approximately 5.5 µg/g of dry weight (17 times
less than the folate content of B. catenulatum ATCC 27539). It is uncommon for humans to consume large
amounts of microbial biomass, except for yeast. Nonetheless, the high specific levels of bifidobacteria and
yeasts can still have a notable impact on fermented foods.

Bifidobacterium animalis subsp. animalis ATCC 25527 is a species in which folate biosynthesis has been
[74]
found absent, with the lowest level per biomass unit corresponding to 2 µg per g DM .

In addition, it is also crucial that the folate-producing strains should be able to grow consistently and in
high amounts under the desired conditions to produce them easily for application. Therefore, the amount of
folate per unit biomass is one of the key factors considered during the screening and development of folate-
producing strains, along with the ease of biomass production and yield obtained from suitable raw