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Page 2 of 15 Geerlings et al. Microbiome Res Rep 2024;3:36 https://dx.doi.org/10.20517/mrr.2024.06

transcriptome and proteome levels, including differences in the expression of glycosyltransferases, signaling
proteins, and stress response. Furthermore, elongated cells and higher OD600 values were observed using the
plant-based media as compared to cultivation media containing mucin.

Conclusion: These differences do not hamper growth, and therefore, our data suggest that the food-grade medium
composition described here could be used to produce A. muciniphila with high yields for therapeutic purposes.

Keywords: A. muciniphila, food-grade medium, human gut microbiota, industrial production

INTRODUCTION
Over the past years, the gut microbiota and its correlation to health and disease have been studied
[1]
extensively . Notably, strong correlations have been made between the gut microbiota composition and
diseases, such as obesity , pre-diabetes , type 2 diabetes, non-alcoholic fatty liver disease and liver
[2,3]
[6]
[4,5]
[7]
cirrhosis . Moreover, a causal involvement of gut microbiota by fecal microbiota transplantation has been
demonstrated in various inflammatory and metabolic diseases . This is increasing the interest in the
[8]
development of interventions aiming to alter the gut microbiota, including those with specific gut bacteria,
also termed next-generation beneficial microbes [9-11] .
While most gut bacteria inhabit the lumen of the colon and thrive on dietary leftovers, Akkermansia
muciniphila (A. muciniphila) is an abundant gut symbiont feeding on the colonic mucosa [12-14] .
A. muciniphila is a Gram-negative bacterium belonging to the Verrucomicrobia phylum, found to be
present in the mucosal layer and specialized in the use of mucin as a single carbon, nitrogen, and energy
source [12,15] . Considerable interest in A. muciniphila derives from human association studies that have
demonstrated an inverse correlation with diabetes and obesity, as well as positive correlations with healthy
metabolic status, as recently reviewed . These results were initially found with deep metagenomic analyses,
[14]
later expanded with species-targeted quantifications, and recently supported by linking thousands of
A. muciniphila metagenomes to host characteristics [3,16,17] .

Direct evidence for the role of A. muciniphila was provided in a series of mouse models where the
[18]
administration of its cells was found to prevent diet-induced obesity . This hallmark study was followed by
many reports showing the beneficial effects of A. muciniphila administration in a variety of mouse
models [19-23] . However, all these studies have been using mucin-based media to cultivate A. muciniphila,
providing a potential bias since this animal-derived glycoprotein is not free from remnants of other bacteria.
A breakthrough came with the development of metabolic models that showed the dependency of
A. muciniphila on exogenously added threonine and its inability to synthesize N-acetylglucosamine
(GlcNAc) from glucose, resulting in synthetic media that obviated the use of mucin by using a combination
of L-threonine, glucose, GlcNAc, and peptone [24,25] . These were used in a series of mechanistic studies in
diabetic and obese mice, demonstrating that pasteurized A. muciniphila cells replicated the beneficial effects
of live cells grown in mucin-free media in diabetic and obese mice . The capacity of pasteurized
[26]
A. muciniphila cells to be at least as effective as live cells was confirmed in a recent clinical trial where their
administration to metabolic syndrome subjects resulted in improved insulin sensitivity, reduced insulinemia
and plasma total cholesterol, and reduction of body weight, including reduced fat mass and hip
[27]
circumference .

Several studies have been focusing on the cultivation, storage, and delivery methods of either pasteurized or
alive A. muciniphila for therapeutic purposes [26,28-31] . Moreover, the environmental conditions in which
A. muciniphila survives have been studied in detail, as it is sensitive to low pH, oxygen and bile salts [12,32,33] .