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Geerlings et al. Microbiome Res Rep 2024;3:36 https://dx.doi.org/10.20517/mrr.2024.06 Page 3 of 15

This information may be useful for the delivery of live A. muciniphila for therapeutic purposes. Along with
the development of synthetic media, the growth characteristics and the physiology of A. muciniphila have
been characterized by comparing mucin growth to that in media containing single sugars, such as glucose
and GlcNAc. This is important as A. muciniphila exerts its health benefits while using mucin as a carbon,
nitrogen, and energy source, and hence, several studies addressed its transcriptome and proteome under
these conditions [24,32] . Recent years have seen an increasing number of signaling molecules that
A. muciniphila is producing, which interact with the host [1,14] . These include the protein Amuc_1100, which
[34]
is known to be part of a set of outer membrane proteins . Preclinical data have shown that the heat-stable
Amuc_1100 protein can reproduce the effects of live and pasteurized cells in protection from diet-induced
obesity and is an efficient ligand for signal transduction to Toll-like receptor 2 (TLR2) [26,35] . Other recently
identified proteins that have been implicated in host signaling include Amuc_1631 (also known as P9),
Amuc_1434, and Amuc_2109, but the location and production of these have not yet been studied [36-39] .

For human interventions or supplementation with A. muciniphila cells, industrial-scale fermentations are
needed and thus the used cultivation media should be not only free from mucin derived from animals but
also food-grade, non-allergenic and enable efficient growth to high densities to provide cost-effective
production platforms. Moreover, to address present consumer needs, such as increasing interest in
flexitarian diets and sustainability, plant-based rather than animal-derived components are to be used.
Finally, in these conditions, there should be sufficient production of signaling molecules that have been
identified as interacting with the host. It is furthermore of importance to assess the safety of A. muciniphila
in the development trajectory for its use in therapeutic applications. A recent study demonstrated the safety
of pasteurized A. muciniphila cells in a variety of in vitro models and a 90-day rat trial . This and other
[40]
information was used by the European Food Safety Authority (EFSA) to approve the use of pasteurized
A. muciniphila cells as a novel food . This all supports the interest in the fermentation optimization of this
[41]
next-generation beneficial microbe, and in this study, we assessed the growth and performance of
A. muciniphila in newly developed food-grade and plant-based media with varying carbon sources using a
[42]
multi-omics approach in comparison with its growth on mucin-containing media .

METHODS
Bacterial strain and culture conditions
The type-strain A. muciniphila Muc (ATCC BAA-835) was used for all cultivation experiments.
T
Basal medium was used in the fermentations for the initial experiments including soy medium and mucin
medium and pre-cultures for the pea peptone bioreactors as described previously [12,43] .

For the initial experiments, soy medium and mucin medium were prepared. To prepare soy medium, 16 g/L
soy peptone (AM41, Organotechnie SAS) was added to basal medium. In addition, GlcNAc and glucose
were added in equimolar amounts to a total of 25 mM (Sigma-Aldrich). Mucin medium was prepared by
adding 0.5% hog gastric mucin (Sigma-Aldrich) to basal medium.

Pre-cultures grown for the anaerobic fermentations supplemented with pea peptone were cultivated in basal
medium supplemented with tryptone (20 g/L) and L-threonine (4 g/L) with the following carbon source
composition: 12.5 mM GlcNAc and 12.5 mM glucose. The cultures were grown in anaerobic conditions and
incubated at 37 °C for 48 h (non-shaking).

Food-grade medium was used for the main experiments with the following composition: KH PO (0.4 g/L),
2
4
Na HPO (0.669 g/L), NH Cl (0.3 g/L), NaCl (0.3 g/L), MgCl 6H O (0.1 g/L), pea peptone A482
4
2
2
2
4