This work aimed to define the microbial consortia that are able to digest gluten into non-toxic and non-immunogenic peptides in the human gastrointestinal tract. Methods: 131 out of 504 tested Bacillus and lactic acid bacteria, specifically Bacillus (64), lactobacilli (63), Pediococcus (1), and Weissella (3), showed strong gastrointestinal resistance and were selected for their PepN, PepI, PepX, PepO, and PepP activities toward synthetic substrates. Based on multivariate analysis, 24 strains were clearly distinct from the other tested strains based on having the highest enzymatic activities. As estimated by RP-HPLC and nano-ESI–MS/MS, 6 cytoplasmic extracts out of 24 selected strains showed the ability to hydrolyze immunogenic epitopes, specifically 57–68 of α9-gliadin, 62–75 of A-gliadin, 134–153 of γ-gliadin, and 57–89 (33-mer) of α2-gliadin. Live and lysed cells of selected strains were combined into different microbial consortia for hydrolyzing gluten under gastrointestinal conditions. Commercial proteolytic enzymes (Aspergillusoryzae E1, Aspergillusniger E2, Bacillussubtilis Veron HPP, and Veron PS proteases) were also added to each microbial consortium. Consortium activity was evaluated by ELISA tests, RP-HPLC-nano-ESI–MS/MS, and duodenal explants from celiac disease patients. Results: two microbial consortia (Consortium 4: Lactiplantibacillus (Lp.) plantarum DSM33363 and DSM33364, Lacticaseibacillus (Lc.) paracasei DSM33373, Bacillussubtilis DSM33298, and Bacilluspumilus DSM33301; and Consortium 16: Lp. plantarum DSM33363 and DSM33364, Lc. paracasei DSM33373, Limosilactobacillusreuteri DSM33374, Bacillusmegaterium DSM33300, B.pumilus DSM33297 and DSM33355), containing commercial enzymes, were able to hydrolyze gluten to non-toxic and non-immunogenic peptides under gastrointestinal conditions. Conclusions: the results of this study provide evidence that selected microbial consortia could potentially improve the digestion of gluten in gluten-sensitive patients by hydrolyzing the immunogenic peptides during gastrointestinal digestion.

Gut microbiota is an important modulator of human health and contributes to high inter-individual variation in response to food and pharmaceutical ingredients. The clinical outcomes of interventions with prebiotics, probiotics, and synbiotics have been mixed and often unpredictable, arguing for novel approaches for developing microbiome-targeted therapeutics. Here, we review how the gut microbiota determines the fate of and individual responses to dietary and xenobiotic compounds via its immense metabolic potential. We highlight that microbial metabolites play a crucial role as targetable mediators in the microbiota-host health relationship. With this in mind, we expand the concept of synbiotics beyond prebiotics' role in facilitating growth and engraftment of probiotics, by focusing on microbial metabolism as a vital mode of action thereof. Consequently, we discuss synbiotic compositions that enable the guided metabolism of dietary or co-formulated ingredients by specific microbes leading to target molecules with beneficial functions. A workflow to develop novel synbiotics is presented, including the selection of promising target metabolites (e.g. equol, urolithin A, spermidine, indole-3 derivatives), identification of suitable substrates and producer strains applying bioinformatic tools, gut models, and eventually human trials.In conclusion, we propose that discovering and enabling specific substrate-microbe interactions is a valuable strategy to rationally design synbiotics that could establish a new category of hybrid nutra-/pharmaceuticals.

The gut microbiota is a crucial modulator of health effects elicited by food components, with SCFA (short chain fatty acids), especially butyrate, acting as important mediators thereof. We therefore developed a nutritional synbiotic composition targeted at shifting microbiome composition and activity towards butyrate production. An intestinal screening model was applied to identify probiotic Bacillus strains plus various amino acids and peptides with suitable effects on microbial butyrate producers and levels. A pilot study was performed to test if the synbiotic formulation could improve fecal butyrate levels in healthy humans. A combination of Bacillus subtilis DSM (Number of German Collection of Microorganisms and Cell Cultures) 32315 plus L-alanyl-L-glutamine resulted in distinctly increased levels of butyrate and butyrate-producing taxa (Clostridium group XIVa, e.g., Faecalibacterium prausnitzii), both in vitro and in humans. Moreover, circulating lipid parameters (LDL-, and total cholesterol and LDL/HDL cholesterol ratio) were significantly decreased and further metabolic effects such as glucose-modulation were observed. Fasting levels of PYY (Peptide YY) and GLP-1 (Glucagon-like Peptide 1) were significantly reduced. In conclusion, our study indicates that this synbiotic composition may provide an effective and safe tool for stimulation of intestinal butyrate production with effects on e.g., lipid and glucose homeostasis. Further investigations in larger cohorts are warranted to confirm and expand these findings.

Specialized pro-resolving mediators (SPM) have emerged as crucial lipid mediators that confer the inflammation-resolving effects of omega-3 polyunsaturated fatty acids (n-3 PUFA). Importantly, SPM biosynthesis is dysfunctional in various conditions, which may explain the inconclusive efficacy data from n-3 PUFA interventions. To overcome the limitations of conventional n-3 PUFA supplementation strategies, we devised a composition enabling the self-sufficient production of SPM in vivo. Bacillus megaterium strains were fed highly bioavailable n-3 PUFA, followed by metabololipidomics analysis and bioinformatic assessment of the microbial genomes. All 48 tested Bacillus megaterium strains fed with the n-3 PUFA formulation produced a broad range of SPM and precursors thereof in a strain-specific manner, which may be explained by the CYP102A1 gene polymorphisms that we detected. A pilot study was performed to test if a synbiotic Bacillus megaterium/n-3 PUFA formulation increases SPM levels in vivo. Supplementation with a synbiotic capsule product led to significantly increased plasma levels of hydroxy-eicosapentaenoic acids (5-HEPE, 15-HEPE, 18-HEPE) and hydroxy-docosahexaenoic acids (4-HDHA, 7-HDHA) as well as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in healthy humans. To the best of our knowledge, we report here for the first time the development and in vivo application of a self-sufficient SPM-producing formulation. Further investigations are warranted to confirm and expand these findings, which may create a new class of n-3 PUFA interventions targeting inflammation resolution.

Beneficial health effects of omega-3 polyunsaturated fatty acids (n-3 PUFA) are partly attributed to specialized pro-resolving mediators (SPMs), which promote inflammation resolution. Strategies to improve n-3 PUFA conversion to SPMs may, therefore, be useful to treat or prevent chronic inflammatory disorders. Here, we explored a synbiotic strategy to increase circulating SPM precursor levels. Healthy participants (n = 72) received either SynΩ3 (250 mg eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA) lysine salts; two billion CFU Bacillus megaterium; n = 23), placebo (n = 24), or fish oil (300 mg EPA plus DHA; N = 25) capsules daily for 28 days in a randomized, double-blind placebo-controlled parallel 3-group design. Biomarkers were assessed at baseline and after 2 and 28 days of intervention. The primary analysis involved the comparison between SynΩ3 and placebo. In addition, SynΩ3 was compared to fish oil. The synbiotic SynΩ3 comprising Bacillus megaterium DSM 32963 and n-3 PUFA salts significantly increased circulating SPM precursor levels, including 18-hydroxy-eicosapentaenoic acid (18-HEPE) plus 5-HEPE, which was not achieved to this extent by fish oil with a similar n-3 PUFA content. Omega-3 indices were increased slightly by both SynΩ3 and fish oil. These findings suggest reconsidering conventional n-3 PUFA supplementation and testing the effectiveness of SynΩ3 particularly in conditions related to inflammation.

Background: Omega-3 polyunsaturated fatty acids (n-3 PUFA) have been used in the treatment of inflammatory bowel diseases (IBD) and irritable bowel syndrome (IBS), and their effects are potentiated upon conversion to specialized pro-resolving mediators (SPM). Recent studies indicated that the probiotic bacterial strain Bacillus megaterium DSM 32963 can be used to enhance the production of SPM and its precursors in vivo. Methods: Here, we explored the contribution of Bacillus megaterium DSM 32963 to SPM production in a validated, dynamic model of the upper and lower intestine. The TIM-1 and TIM-2 models were applied, with the TIM-2 model inoculated with the fecal microbiota of healthy individuals and probed with an n-3 PUFA lysine salt with and without Bacillus megaterium DSM 32963 or an SPM-enriched fish oil or placebo. Kinetics of SPM production were assessed by metabololipidomics analysis, and survival and engraftment of the Bacillus megaterium strain was monitored by plate counting and by strain-specific qPCR. Results: Bacillus megaterium DSM 32963 poorly survived TIM-1 conditions but propagated in the TIM-2 model, where it enabled the metabolism of n-3 PUFA to SPM (resolvin E2 and protectin DX) and SPM precursors (e.g., 5-hydroxyeicosapentaenoic acid (5-HEPE), 15-HEPE, 18-HEPE, 4-hydroxydocosahexaenoic acid (4-HDHA), 10-HDHA, and 17-HDHA, among other EPA- and DHA-derived metabolites) with significantly higher levels of lipid mediator production compared to the n-3 PUFA lysine salt alone; esterified n-3 PUFA were hardly converted by the microbiota. Conclusions: These findings reinforce that Bacillus megaterium DSM 32963 facilitates SPM production in situ from bioavailable n-3 PUFA in the large intestine, highlighting its use to complement eukaryotic SPM biosynthesis by the host and its possible therapeutic use for, e.g., IBD and IBS.