Selection of Gut-Resistant Bacteria and Construction of Microbial Consortia for Improving Gluten Digestion under Simulated Gastrointestinal Conditions

Abstract

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.

Keywords: Bacillus; Lacticaseibacillus; Lactiplantibacillus; Limosilactobacillus; bacterial peptidases; gluten epitopes.

Figure 1

Viable cell count (CFU/mL) of the bacterial strains tested for their resistance to the gastro (G) and then intestinal (I) transit in vitro at different conditions: pH 2 (pH2), pH 3 (pH3), pH 2 plus skim milk (pH2SM), and pH 8 (pH8). All strains belong to Bacillus, lactobacilli, Weissella, Leuconostoc, and Pediococcus species. The group named “other lactic acid bacteria” includes Leuconostoc and Pediococcus strains.

Figure 2

Score (A) and loading (B) plots of the first and second principal components (PC) after principal component analysis (PCA) based on the general aminopeptidase type N (PepN), proline iminopeptidase (PepI), X-prolyl dipeptidyl aminopeptidase (PepX), endopeptidase (PepO), and prolyl endopeptidase (PepP) activities of the cytoplasmic extracts of the 131 Bacillus, lactobacilli, and Weissella strains. PepN, PepI, PepX, and PepP were measured using Leu-p-nitroanilides (p-NA), Pro-p-NA, Gly-Pro-p-NA, Z-Gly-Gly-Leu-p-NA, and Z-Gly-Pro-4-nitroanilide substrates, respectively. All strains are reported in the plot by circles and the strain code was reported only for the selected strains.

Figure 3

The tested bacterial cytoplasmic extracts (CE1–CE6) and the related peptidase activities against immunogenic epitopes.

Figure 4

Concentration (ng/µL) of interleukin 2 (IL-2) (A), interleukin 10 (IL-10) (B), and interferon gamma (IFN-γ) (C) in duodenal biopsy specimens from patients with CD. Analyses were carried out in two independent experiments. Control, wheat bread digested without the addition of bacterial cells and microbial enzymes; RPMI+gastric and intestinal juice, negative control; Consortium 4, wheat bread digested with the addition of live and lysed cells of Lp. plantarum DSM33363 and DSM33364, Lc. paracasei DSM33373, B. subtilis DSM33298, and B. pumilus DSM33301 and E1, E2, Veron PS, and Veron HPP commercial enzymes); Consortium 7, wheat bread digested with the addition of live and lysed cells of Lp. plantarum DSM33362, DSM33366, and DSM33370, Ls. reuteri DSM33374, B. megaterium DSM33356, and B. subtilis DSM33353 and E1, E2, Veron PS, and Veron HPP commercial enzymes; and Consortium 16, wheat bread digested with the addition of live and lysed cells of Lp. plantarum DSM33363 and DSM33364, Lc. paracasei DSM33373, Ls. reuteri DSM33374, B. megaterium DSM33300, B. pumilus DSM33297 and DSM33355 and E1, E2, Veron PS, and Veron HPP commercial enzymes. CD1 to CD10, duodenal biopsy specimens from celiac patients. The evaluation was conducted on biological duplicates and the mean values (± standard deviation) were reported.