Functional Muffins Exert Bifidogenic Effects along with Highly Product-Specific Effects on the Human Gut Microbiota Ex Vivo

Abstract

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.

Keywords: berry blast muffin (BBM); functional foods; lemon chia muffin (LCM); oat spice mookie (OSM); prebiotics; probiotics; short-chain fatty acid (SCFA); systemic intestinal fermentation research (SIFR).

Figure 1

Schematic overview of the study design using ex vivo SIFR^® technology. (a) Reactor design using the ex vivo SIFR^® technology to evaluate the impact of GoodBiome^TM Foods against an unsupplemented parallel control (NSC = no substrate control). (b) Timeline and analysis at different timepoints.

Figure 2

The fecal microbiota covered clinically relevant interpersonal differences. Abundances (%) of the key families (top 15), as quantified via shallow shotgun sequencing, in the fecal microbiota of each of the four human adults that provided a fecal donation for the current SIFR^® study.

Figure 3

GoodBiome™ Foods exerted marked effects on microbial metabolic activity over time. The effects on (A) pH, (B) gas production, (C) total SCFA, (D) acetate, (E) propionate, (F) butyrate, (G) valerate, and (H) bCFA were compared for GoodBiome™ Foods versus an unsupplemented control (NSC) at 6 h, 24 h, 30 h, and 48 h after the initiation of colonic incubation. Data were presented as means across simulations for four individual donors (n = 3 per donor). The statistical significance of the treatment effects for the test products vs. NSC within each timepoint can be found in Figures S2 and S3.

Figure 4

GoodBiome™ Foods exerted significant impact on microbial composition at phylum level. Samples were collected 30 h after the colonic incubations were initiated. Data were expressed as average absolute levels (cells/mL) of each phylum across simulations for four individual donors (n = 3 per donor). The statistical significance of the potential treatment effects within each comparison was determined via Benjamani–Hochberg post hoc testing. Significant changes (p_adjusted < 0.05) were indicated with asterisks.

Figure 5

GoodBiome™ Foods exerted significant impact on microbial composition at species level. The bar charts were generated for species that were significantly (FDR = 0.05) affected by any of the treatments at 30 h, expressed as log2fold change (treatment/NSC), averaged across four human adults (n = 3 per donor). Purple and red bars indicated significant/consistent decreases and increases, respectively. Notable health- or disease-related taxa are highlighted in a gray box.

Figure 6

The GoodBiome™ Foods exerted significant impact on taxa that are potentially relevant for human health. Violin plots, expressed as log2fold change (treatment/NSC), were presented for four individual human adults (n = 3). The data were presented for (A) Clostridiodes difficile (B) Bifidobacteriaceae, (C) Anaerobutyricum hallii, (D) Bacteroidaceae, Bacteroidales_u_f, and Tannerellaceae. For (B–D), Pearson correlation analysis demonstrated significant positive correlations (p < 0.05) between the absolute levels of these taxa (cells/mL) and the concentration (mM) of the most relevant SCFA related to these taxa, i.e., (A) acetate, (B) butyrate, and (C) propionate.

Figure 7

The GoodBiome™ Foods exerted significant impact on the production of microbial metabolites, well beyond SCFA. The bars were generated for metabolites that were significantly (FDR = 0.05) affected by any of the treatments, expressed as log2fold change (treatment/NSC), averaged across four human adults (n = 3 per test subject). Purple and red bars indicated significant decreases and increases, respectively.