Background: The human gut microbiota develops in concordance with its host over a lifetime, resulting in age-related shifts in community structure and metabolic function. Little is known about whether these changes impact the community’s response to microbiome-targeted therapeutics. Providing critical information on this subject, faecal microbiomes of subjects from six age groups, spanning from infancy to 70-year-old adults (n = six per age group) were harvested. The responses of these divergent communities to treatment with the human milk oligosaccharide 2’-fucosyllactose (2’FL), fructo-oligosaccharides (FOS), and lactose was investigated using the Ex vivo SIFR^® technology that employs bioreactor fermentation and is validated to be predictive of clinical findings. Additionally, it was evaluated whether combining faecal microbiomes of a given age group into a single pooled microbiome produced similar results as the individual microbiomes. Results: First, marked age-dependent changes in community structure were identified. Bifidobacterium levels strongly declined as age increased, and Bifidobacterium species composition was age-dependent: B. longum, B. catenulatum/pseudocatenulatum, and B. adolescentis were most prevalent for breastfed infants, toddlers/children, and adults, respectively. Metabolomic analyses (LA-REIMS) demonstrated that these age-dependent differences particularly impacted treatment effects of 2’FL (more than FOS/lactose). Further analysis revealed that while 2’FL enhanced production of short-chain fatty acids (SCFAs) and exerted potent bifidogenic effects, regardless of age, the specific Bifidobacterium species enhanced by 2’FL, as well as subsequent cross-feeding interactions, were highly age-dependent. Furthermore, single-pooled microbiomes produced results that were indicative of the average treatment response for each age group. Nevertheless, pooled microbiomes had an artificially high diversity, thus overestimating treatment responses (especially for infants), did not recapitulate interindividual variation, and disallowed for the correlative analysis required to unravel mechanistic actions. Conclusions: Age is an important factor in shaping the gut microbiome, with the dominant taxa and their metabolites changing over a lifetime. This divergence affects the response of the microbiota to therapeutics, demonstrated in this study using 2’FL. These results evidence the importance of screening across multiple age groups separately to provide granularity of how therapeutics impact the microbiome and, consequently, human health.

Cow’s milk protein allergy (CMPA) in infancy is associated with intestinal microbial dysbiosis, characterised by low Bifidobacteriaceae levels. The present study aimed to investigate the impact of two human milk oligosaccharides (HMO), lactose (L), and their combination on the faecal microbiome and metabolome of infants with CMPA. Stool samples of 12 term infants with probable CMPA (mean age 4.3 months) were analysed using a validated intestinal fermentation assay (SIFR^® technology). For each substrate (i.e. HMO (2′-fucosyllactose [2′-FL] and lacto-N-neotetraose [LNnT]), L and HMO + L), taxonomic microbiome characterisation and untargeted metabolite profiling were performed at multiple timepoints. At baseline, the tested faecal microbiota overall displayed low abundances of Bifidobacteriaceae. Fermentation with either HMO or lactose significantly enriched Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium pseudocatenulatum and, for HMO + L, also Bifidobacterium bifidum. The increase in HMO-utilising bifidobacteria was associated with a significant rise in levels of short-chain fatty acids, aromatic lactic acids and N-acetylated amino acids, with additive effects being observed for HMO + L. The above data suggest that the combination of 2′-FL, LNnT and lactose helps to alleviate the previously reported CMPA-associated intestinal bacterial dysbiosis and induces the production of several beneficial metabolites. The clinical significance of these findings for infants with CMPA requires further investigation.

GoodBiome™ Foods are functional foods containing a probiotic (Bacillus subtilis HU58™) and prebiotics (mainly inulin). Their effects on the human gut microbiota were assessed using ex vivo SIFR^® technology, which has been validated to provide clinically predictive insights. GoodBiome™ Foods (BBM/LCM/OSM) were subjected to oral, gastric, and small intestinal digestion/absorption, after which their impact on the gut microbiome of four adults was assessed (n = 3). All GoodBiome™ Foods boosted health-related SCFA acetate (+13.1/14.1/13.8 mM for BBM/LCM/OSM), propionate (particularly OSM; +7.4/7.5/8.9 mM for BBM/LCM/OSM) and butyrate (particularly BBM; +2.6/2.1/1.4 mM for BBM/LCM/OSM). This is related to the increase in Bifidobacterium species (B. catenulatum, B. adolescentis, B. pseudocatenulatum), Coprococcus catus and Bacteroidetes members (Bacteroides caccae, Phocaeicola dorei, P. massiliensis), likely mediated via inulin. Further, the potent propionogenic potential of OSM related to increased Bacteroidetes members known to ferment oats (s key ingredient of OSM), while the butyrogenic potential of BBM related to a specific increase in Anaerobutyricum hallii, a butyrate producer specialized in the fermentation of erythritol (key ingredient of BBM). In addition, OSM/BBM suppressed the pathogen Clostridioides difficile, potentially due to inclusion of HU58™ in GoodBiome™ Foods. Finally, all products enhanced a spectrum of metabolites well beyond SCFA, including vitamins (B3/B6), essential amino acids, and health-related metabolites such as indole-3-propionic acid. Overall, the addition of specific ingredients to complex foods was shown to specifically modulate the gut microbiome, potentially contributing to health benefits. Noticeably, our findings contradict a recent in vitro study, underscoring the critical role of employing a physiologically relevant digestion/absorption procedure for a more accurate evaluation of the microbiome-modulating potential of complex foods.

The food additive sodium benzoate (SB) has been used for decades as an antimicrobial to prevent food spoilage. SB has been deemed to pose no risk to human health when consumed at levels under 5 mg/kg body weight per day; however, when many of the supporting studies were conducted, the importance of the gut microbiome to human health was not yet appreciated. Given SB’s known antimicrobial qualities, it is important to assess the effect of this food additive on the human gut microbiome. The ex vivo SIFR^® (Systemic Intestinal Fermentation Research) technology was used to test the effect of SB on microbial communities from 24 donors, aged infants to older adults. A dose of 3.5 g/L SB elicited a drop in the Pseudomonadota phylum for multiple age groups but did not alter the alpha or beta diversity within any of these groups. This was accompanied by changes in the functional outputs that included an overall rise in butyrate and a drop in propionate production. This higher butyrate correlates with an increase in the abundance of several known butyrate producers in the presence of SB, although the genetic potential for its production in the community did not change. Overall, despite using a dose ten times higher than the accepted daily intake limit, the effect on the gut microbiome was minimal.

Hydrogen sulfide (H₂S) can be produced from the metabolism of foods containing sulfur in the gastrointestinal tract (GIT). At low doses, H₂S regulates the gut microbial community and supports GIT health, but depending on dose, age, and individual health conditions, it may also contribute to inflammatory responses and gut barrier dysfunction. Controlling H₂S production in the GIT is important for maintaining a healthy gut microbiome. However, research on this subject is limited due to the gaseous nature of the chemical and the difficulty of accessing the GIT in situ. In the present ex vivo experiment, we used a single-dose sodium sulfide preparation (SSP) as a H₂S precursor to test the effect of H₂S on the human gut microbiome across different age groups, including breastfed infants, toddlers, adults, and older adults. Metagenomic sequencing and metabolite measurements revealed that the development of the gut microbial community and the production of short-chain fatty-acids (SCFAs) were age-dependent; that the infant and the older adult groups were more sensitive to SSP exposure; that exogeneous SSP suppressed SCFA production across all age groups, except for butyrate in the older adult group, suggesting that H₂S selectively favors specific gut microbial processes.