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

Maria De Angelis, Sonya Siragusa, Mirco Vacca, Raffaella Di Cagno, Fernanda Cristofori, Michael Schwarm, Stefan Pelzer, Monika Flügel, Bodo Speckmann, Ruggiero Francavilla, Marco Gobbetti, Maria De Angelis, Sonya Siragusa, Mirco Vacca, Raffaella Di Cagno, Fernanda Cristofori, Michael Schwarm, Stefan Pelzer, Monika Flügel, Bodo Speckmann, Ruggiero Francavilla, Marco Gobbetti

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.

Conflict of interest statement

M.S., S.P., M.F., and B.S. declare competing interests as employees of Evonik Operations GmbH. Other authors declare no conflict of interest.

Figures

Figure 1
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
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
Figure 3
The tested bacterial cytoplasmic extracts (CE1–CE6) and the related peptidase activities against immunogenic epitopes.
Figure 4
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.

References

    1. Biesiekierski J.R. What is gluten? J. Gastroenterol. Hepatol. 2017;32:78–81. doi: 10.1111/jgh.13703.
    1. Khan H. Genetic improvement for end-use quality in wheat. In: Qureshi A., Dar Z., Wani S., editors. Quality Breeding in Field Crops. Springer; Cham, Switzerland: 2019. pp. 239–253.
    1. Shewry P.R., Halford N.G., Lafiandra D. Genetics of wheat gluten proteins. In: Hall J.C., Dunlap J.C., Friedmann T., editors. Advances in Genetics. Volume 49. Academic Press; Cambridge, MA, USA: 2003. pp. 111–184.
    1. Arentz–Hansen H., Mcadam S.N., Molberg Ø., Fleckenstein B., Lundin K.E., Jørgensen T.J., Jung G., Roepstorff P., Sollid L.M. Celiac lesion T cells recognize epitopes that cluster in regions of gliadins rich in proline residues. Gastroenterology. 2002;123:803–809. doi: 10.1053/gast.2002.35381.
    1. Barro Losada F., Lehisa J., Giménez M.J., García-Molina M.D., Ozuna C., Comino I., Sousa Martín C., Gil Humanes J. Targeting of prolamins by RNAi in bread wheat: Effectiveness of seven silencing-fragment combinations for obtaining lines devoid of coeliac disease epitopes from highly immunogenic gliadins. Plant Biotechnol. J. 2016;14:986–996. doi: 10.1111/pbi.12455.
    1. Dewar D.H., Amato M., Ellis H.J., Pollock E.L., Gonzalez-Cinca N., Wieser H., Ciclitira P.J. The toxicity of high molecular weight glutenin subunits of wheat to patients with coeliac disease. Eur. J. Gastroenterol. Hepatol. 2006;18:483–491. doi: 10.1097/00042737-200605000-00005.
    1. Stamnaes J., Sollid L.M. Seminars in Immunology. Volume 27. Academic Press; Cambridge, MA, USA: 2015. Celiac disease: Autoimmunity in response to food antigen; pp. 343–352.
    1. Potter M.D., Walker M.M., Keely S., Talley N.J. What’s in a name? ‘Non-coeliac gluten or wheat sensitivity’: Controversies and mechanisms related to wheat and gluten causing gastrointestinal symptoms or disease. Gut. 2018;67:2073–2077. doi: 10.1136/gutjnl-2018-316360.
    1. D’argenio V., Casaburi G., Precone V., Pagliuca C., Colicchio R., Sarnataro D., Discepolo V., Kim S.M., Russo I., Del Vecchio Blanco G., et al. Metagenomics reveals dysbiosis and a potentially pathogenic N. flavescens strain in duodenum of adult celiac patients. Am. J. Gastroenterol. 2016;111:879. doi: 10.1038/ajg.2016.95.
    1. Wacklin P., Laurikka P., Lindfors K., Collin P., Salmi T., Lähdeaho M.L., Saavalainen O., Mäki M., Mättö J., Kurppa K., et al. Altered duodenal microbiota composition in celiac disease patients suffering from persistent symptoms on a long-term gluten-free diet. Am. J. Gastroenterol. 2014;109:1933–1941. doi: 10.1038/ajg.2014.355.
    1. Niland B., Cash B.D. Health benefits and adverse effects of a gluten-free diet in non–celiac disease patients. Gastroenterol. Hepatol. 2018;14:82.
    1. Larretxi I., Simon E., Benjumea L., Miranda J., Bustamante M.A., Lasa A., Churruca I. Gluten-free-rendered products contribute to imbalanced diets in children and adolescents with celiac disease. Eur. J. Nutr. 2018;58:775–783. doi: 10.1007/s00394-018-1685-2.
    1. Wild D., Robins G.G., Burley V.J., Howdle P.D. Evidence of high sugar intake, and low fibre and mineral intake, in the gluten-free diet. Aliment. Pharmacol. Ther. 2010;32:573–581. doi: 10.1111/j.1365-2036.2010.04386.x.
    1. Herrán A.R., Pérez-Andrés J., Caminero A., Nistal E., Vivas S., de Morales J.M.R., Casqueiro J. Gluten-degrading bacteria are present in the human small intestine of healthy volunteers and celiac patients. Res. Microbiol. 2017;168:673–684. doi: 10.1016/j.resmic.2017.04.008.
    1. Gobbetti M., Pontonio E., Filannino P., Rizzello C.G., De Angelis M., Di Cagno R. How to improve the gluten-free diet: The state of the art from a food science perspective. Food Res. Int. 2018;110:22–32. doi: 10.1016/j.foodres.2017.04.010.
    1. Ribeiro M., Nunes F.M., Rodriguez-Quijano M., Carrillo J.M., Branlard G., Igrejas G. Next-generation therapies for celiac disease: The gluten-targeted approaches. Trends Food Sci. Technol. 2018;75:56–71. doi: 10.1016/j.tifs.2018.02.021.
    1. Francavilla R., Piccolo M., Francavilla A., Polimeno L., Semeraro F., Cristofori F., Castellaneta S., Barone M., Indrio F., Gobbetti M., et al. Clinical and microbiological effect of a multispecies probiotic supplementation in celiac patients with persistent IBS-type symptoms: A randomized, double-blind, placebo-controlled, multicenter trial. J. Clin. Gastroenterol. 2019;53:e117. doi: 10.1097/MCG.0000000000001023.
    1. Cavaletti L., Taravella A., Carrano L., Carenzi G., Sigurtà A., Solinas N., De Caro S., Di Stasio L., Picascia S., Laezza M., et al. E40, a novel microbial protease efficiently detoxifying gluten proteins, for the dietary management of gluten intolerance. Sci. Rep. 2019;9:1–11. doi: 10.1038/s41598-019-48299-7.
    1. Serena G., Kelly C.P., Fasano A. Nondietary therapies for celiac disease. Gastroenterol. Clin. N. Am. 2019;48:145–163. doi: 10.1016/j.gtc.2018.09.011.
    1. Caputo I., Lepretti M., Martucciello S., Esposito C. Enzymatic strategies to detoxify gluten: Implications for celiac disease. Enzym. Res. 2010;2010:174354. doi: 10.4061/2010/174354.
    1. de Sousa Moraes L.F., Grzeskowiak L.M., de Sales Teixeira T.F., Peluzio M.D.C.G. Intestinal microbiota and probiotics in celiac disease. Clin. Microbiol. Rev. 2014;27:482–489. doi: 10.1128/CMR.00106-13.
    1. Plugis N.M., Khosla C. Therapeutic approaches for celiac disease. Best Pract. Res. Clin. Gastroenterol. 2015;29:503–521. doi: 10.1016/j.bpg.2015.04.005.
    1. Francavilla R., Cristofori F., Vacca M., Barone M., De Angelis M. Advances in understanding the potential therapeutic applications of gut microbiota and probiotic mediated therapies in celiac disease. Expert Rev. Gastroenterol. Hepatol. 2020;14:323–333. doi: 10.1080/17474124.2020.1745630.
    1. Mickowska B., Socha P., Urminská D. Immunochemical evaluation of proteolysis of cereal proteins causing celiac disease by microbial proteases. Food Agric. Immunol. 2016;27:743–757. doi: 10.1080/09540105.2016.1148665.
    1. Krishnareddy S., Stier K., Recanati M., Lebwohl B., Green P.H. Commercially available glutenases: A potential hazard in coeliac disease. Ther. Adv. Gastroenterol. 2017;10:473–481. doi: 10.1177/1756283X17690991.
    1. Francavilla R., De Angelis M., Rizzello C.G., Cavallo N., Dal Bello F., Gobbetti M. Selected probiotic lactobacilli have the capacity to hydrolyze gluten peptides during simulated gastrointestinal digestion. Appl. Environ. Microbiol. 2017;83 doi: 10.1128/AEM.00376-17.
    1. Fernández M.F., Boris S., Barbes C. Probiotic properties of human lactobacilli strains to be used in the gastrointestinal tract. J. Appl. Microbiol. 2003;94:449–455. doi: 10.1046/j.1365-2672.2003.01850.x.
    1. Zárate G., Chaia A.P., González S., Oliver G. Viability and beta-galactosidase activity of dairy propionibacteria subjected to digestion by artificial gastric and intestinal fluids. J. Food Prot. 2000;63:1214–1221. doi: 10.4315/0362-028X-63.9.1214.
    1. De Angelis M., Siragusa S., Berloco M., Caputo L., Settanni L., Alfonsi G., Amerio M., Grandi A., Ragni A., Gobbetti M. Selection of potential probiotic lactobacilli from pig feces to be used as additives in pelleted feeding. Res. Microbiol. 2006;157:792–801. doi: 10.1016/j.resmic.2006.05.003.
    1. De Angelis M., Cassone A., Rizzello C.G., Gagliardi F., Minervini F., Calasso M., Di Cagno R., Francavilla R., Gobbetti M. Mechanism of degradation of immunogenic gluten epitopes from Triticum turgidum L. var. durum by sourdough lactobacilli and fungal proteases. Appl. Environ. Microbiol. 2010;76:508–518. doi: 10.1128/AEM.01630-09.
    1. Walker-Smith J.A.G.S. Revised criteria for diagnosis of celiac disease. Arch. Dis. Child. 1990;65:909–911.
    1. Browning T.H., Trier J.S. Organ culture of mucosal biopsies of human small intestine. J. Clin. Investig. 1969;48:1423–1432. doi: 10.1172/JCI106108.
    1. De Angelis M., Rizzello C.G., Fasano A., Clemente M.G., De Simone C., Silano M., De Vincenzi M., Losito I., Gobbetti M. VSL#3 probiotic preparation has the capacity to hydrolyze gliadin polypeptides responsible for celiac sprue probiotics and gluten intolerance. Biochim. Biophys. Acta (BBA) Mol. Basis Dis. 2006;1762:80–93. doi: 10.1016/j.bbadis.2005.09.008.
    1. Rizzello C.G., De Angelis M., Di Cagno R., Camarca A., Silano M., Losito I., De Vincenzi M., De Bari M.D., Palmisano F., Maurano F., et al. Highly efficient gluten degradation by lactobacilli and fungal proteases during food processing: New perspectives for celiac disease. Appl. Environ. Microbiol. 2007;73:4499–4507. doi: 10.1128/AEM.00260-07.
    1. Smecuol E., Hwang H.J., Sugai E., Corso L., Chernavsky A.C., Bellavite F.P., González A., Vodánovich F., Moreno M.L., Vázquez H., et al. Exploratory, randomized, double-blind, placebo-controlled study on the effects of Bifidobacterium infantis natren life start strain super strain in active celiac disease. J. Clin. Gastroenterol. 2013;47:139–147. doi: 10.1097/MCG.0b013e31827759ac.
    1. Sarno M., Lania G., Cuomo M., Nigro F., Passannanti F., Budelli A., Fasano F., Troncone R., Auricchio S., Barone M.V., et al. Lactobacillus paracasei CBA L74 interferes with gliadin peptides entrance in Caco-2 cells. Int. J. Food Sci. Nutr. 2014;65:953–959. doi: 10.3109/09637486.2014.940283.
    1. Giorgi A., Cerrone R., Capobianco D., Filardo S., Mancini P., Zanni F., Fanelli S., Mastromarino P., Mosca L. A probiotic preparation hydrolyzes gliadin and protects intestinal cells from the toxicity of pro-inflammatory peptides. Nutrients. 2020;12:495. doi: 10.3390/nu12020495.
    1. Laparra J.M., Sanz Y. Bifidobacteria inhibit the inflammatory response induced by gliadins in intestinal epithelial cells via modifications of toxic peptide generation during digestion. J. Cell Biochem. 2010;109:801–807. doi: 10.1002/jcb.22459.
    1. Lindfors K., Blomqvist T., Juuti-Uusitalo K., Stenman S., Venäläinen J., Mäki M., Kaukinen K. Live probiotic Bifidobacterium lactis bacteria inhibit the toxic effects induced by wheat gliadin in epithelial cell culture. Clin. Exp. Immunol. 2008;152:552–558. doi: 10.1111/j.1365-2249.2008.03635.x.
    1. Caminero A., Herrán A.R., Nistal E., Pérez-Andrés J., Vaquero L., Vivas S., Ruiz de Morales J.M.G., Albillos S.M., Casqueiro J. Diversity of the cultivable human gut microbiome involved in gluten metabolism: Isolation of microorganisms with potential interest for coeliac disease. FEMS Microbiol. Ecol. 2014;88:309–319. doi: 10.1111/1574-6941.12295.
    1. Caminero A., Galipeau H.J., McCarville J.L., Johnston C.W., Bernier S.P., Russell A.K., Jury J., Herran A.R., Casqueiro J., Tye-Din J.A., et al. Duodenal bacteria from patients with celiac disease and healthy subjects distinctly affect gluten breakdown and immunogenicity. Gastroenterology. 2016;151:670–683. doi: 10.1053/j.gastro.2016.06.041.
    1. Socha P., Mickowska B., Urminská D., Kačmárová K. The use of different proteases to hydrolyze gliadins. J. Microbiol. Biotechnol. Food Sci. 2020;9:101–104. doi: 10.15414/jmbfs.2015.4.special2.101-104.
    1. Shan L., Marti T., Sollid L.M., Gray G.M., Khosla C. Comparative biochemical analysis of three bacterial prolyl endopeptidases: Implications for coeliac sprue. Biochem. J. 2004;383:311–318. doi: 10.1042/BJ20040907.
    1. Gobbetti M., De Angelis M., Di Cagno R., Calasso M., Archetti G., Rizzello C.G. Novel insights on the functional/nutritional features of the sourdough fermentation. Int. J. Food Microbiol. 2019;302:103–113. doi: 10.1016/j.ijfoodmicro.2018.05.018.
    1. Kontakou M., Przemioslo R.T., Sturgess R.P., Limb G.A., Ellis H.J., Day P., Ciclitira P.J. Cytokine mRNA expression in the mucosa of treated coeliac patients after wheat peptide challenge. Gut. 1995;37:52–57. doi: 10.1136/gut.37.1.52.
    1. Goel G., Tye-Din J.A., Qiao S.W., Russell A.K., Mayassi T., Ciszewski C., Sarna V.K., Wang S., Goldstein K.E., Dzuris J.L., et al. Cytokine release and gastrointestinal symptoms after gluten challenge in celiac disease. Sci. Adv. 2019;5:eaaw7756. doi: 10.1126/sciadv.aaw7756.
    1. Makharia G.K.D. Current and emerging therapy for celiac disease. Front. Med. 2014;1:6. doi: 10.3389/fmed.2014.00006.
    1. Freitag T.L., Rietdijk S., Junker Y., Popov Y., Bhan A.K., Kelly C.P., Terhorst C., Schuppan D. Gliadin-primed CD4+ CD45RBlowCD25− T cells drive gluten-dependent small intestinal damage after adoptive transfer into lymphopenic mice. Gut. 2009;58:1597–1605. doi: 10.1136/gut.2009.186361.
    1. Freitag T.L., Loponen J., Messing M., Zevallos V., Andersson L.C., Sontag-Strohm T., Saavalainen P., Schuppan D., Salovaara H., Meri S. Testing safety of germinated rye sourdough in a celiac disease model based on the adoptive transfer of prolamin-primed memory T cells into lymphopenic mice. Am. J. Physiol. Gastrointest. Liver. Physiol. 2014;306:G526–G534. doi: 10.1152/ajpgi.00136.2013.

Source: PubMed

Szponzorok és közreműködők

Egészségi állapot

Kábítószer-beavatkozások

3
Iratkozz fel