Esposito, Alessia (2025) Development of a synbiotic dietary supplement containing potential Next- Generation Probiotics for the modulation of the gut microbiome and metabolome. [Tesi di dottorato]

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Abstract

The term Next-Generation Probiotics (NGPs) usually applies to microbial strains that can have a positive effect on human health, but do not belong to common probiotic species (e.g., lactic acid bacteria, Bifidobacterium). Indeed, large metagenomics surveys of the human gut microbiome carried out in the past years led to the discovery of a number of previously unexplored microorganisms and highlighted their potential role in promoting health. Among the most interesting species proposed as potential NGPs there are Roseburia intestinalis, Eubacterium spp., Ruminococcus bromii, Akkermansia muciniphila, Faecalibacterium prausnitzii, Clostridium spp. and Bacteroides spp.. A feature of some NGPs is the production ofshort chain fatty acids, of whichparticularly butyrate is known to support immune homeostasis and human intestinal health. Dominant butyrate producers in the human gut include A. muciniphila, F. prausnitzii, Ruminococcus bromii, as well as Clostridium, Bacteroides, Roseburia, and Eubacterium species. The large group of butyrate producing gut commensals is thus phylogenetically more diverse than the conventional probiotic lactic acid bacteria, and are reported to confer a large variety of beneficial and therapeutic effects. Akkermansia, which is able to grow on mucin as the sole source of carbon and nitrogen, is generally known for its association with a healthymucosaandimmunemodulatingproperties. Additionally,thebacterium has been shown to prevent high fat diet-induced obesity in mice, improve immunotherapy effects against epithelial tumors in mice transplanted with human microbiota and enhance therapeutic drug effects against murine type 2 diabetes. Members of the Clostridium clusters IV, XIVa and XVIII, which all lack virulence factors, are also important immune homeostasis regulators and observations of immune cell induction suggest that they work as therapeutic agents against gut inflammatory disorders. Additionally, F. prausnitzii from cluster IV has been reported to ameliorate gut inflammation in colitis patients. More recently, the gut commensal Christensenella minuta, which is associated with a lean body type in humans, has gained attention for mitigating lifestyle induced obesity and IBD. However, in most of the cases, we only have results from observational studies, where positive associations of these taxa with health-related parameters was found in gut metagenomes. Indeed, to accelerate the path towards the use of NGPs, more in vitro and in vivo studies would be necessary to explore the effective production of the beneficial metabolites produced. These microbes are challenging to work with due to their extreme sensitivity towards oxygen and often also to gastric conditions encountered after ingestion. The difficulty in maintaining viability of these sensitive bacteria during common preparation, storage and delivery methods challenges the development of NGP into commercial products. Thanks to the evolution in cultivation techniques, fastidious anaerobic microbes from the gut are increasingly being cultured and identified. Another issue is related to the huge strain-level diversity existing within these species: most of our knowledge is limited to the species-level resolution, while wide genomic studies have highlighted that different strains within each species may have potentially divergent activities and possibly a contrasting behavior in relationship with human health. For example, in thegenus Ruminococcus,that is dominant in the human gut, different species, namely R. gnavus, R. torques, and R. bromii, have been linked with health or disease. R. torques and R. gnavus have been linked with gut inflammation, inflammatory bowel disease (IBD), and irritable bowel syndrome, allergy, and early stages of colorectal cancer (CRC), whereas Ruminococcus bromii has been identified as a promising NGP thanks to its ability to hydrolyze resistant starch. NGPs may explicate their positive impact on human health through the production of beneficial metabolites. Short-chain fatty acids (SCFAs) from fiber fermentation are the most studied, since they confer well-established health effects, such as the support to immune homeostasis and intestinal health, the anti-inflammatory, anti-obesity, anti-diabetes, anticancer, cardiovascular protective, hepatoprotective, and neuroprotective activities. However, other bioactive compounds may be also produced, such as vitamins, neuroactive molecules (e.g., serotonin, indoles), and compounds coming from polyphenols catabolism (e.g., urolithins, equol), all of which have a major role in the regulation of the physiology of the host. Since NGP taxa do not have an history of safe use, they will be likely incorporated into the drug regulatory framework, although legislation in Europe is still not clear. However, few years ago Akkermansia muciniphila was firstly approved for the use in the pasteurized form as novel food (pursuant to Regulation (EU) 2015/2283). This further boosted the industrial and academic research on NGPs. Indeed, there is a growing interest in the development of strain collections of indigenous gut microbes, to be commercially exploited as soon as the EU regulation will allow. Besides legislative uncertainties, these taxa are challenging to work with, due to their demanding nutritional requirements, their extreme sensitivity towards oxygen and often also to gastric conditions encountered after ingestion. For these reasons, several efforts are still necessary in order to optimize their cultivation conditions, maintaining their viability during common preparation, storage and delivery methods. In this study, we isolated and characterized potential strains of NGPs for the production of beneficial metabolites, both in vitro and in a Simulator of Human Intestinal Microbial Ecosystem (SHIME) model. In particular, Chapter 2 provides an introduction to the intestinal microbiome and its function; Chapter 3 gives an overview about NGPs strains and their features; in Chapter 4 the screening and selection of suitable culture media for strain isolation from the human gut is described; Chapter 5 focuses on the isolation of anaerobic strains from the human gut of subjects consuming a diet rich in fibre; in Chapter 6 strain in vitro characterization is carried out, describing genomic in silico analysis, phenotypic tests (screening for growth in presence and absence of oxygen, ability to use different complex polysaccharides, in vitro production of beneficial metabolites, such as short-chain fatty acids from carbohydrates, urolithins and equol from polyphenols); finally, in Chapter 7, the best performing strains have been selected for the production of a synbiotic supplement and tested in SHIME model to evaluate the impact on the gut microbiome.

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