A method to identify a consortium of probiotic strains suitable for gluten degradation

Abstract

The present invention relates to a method for identifying a consortium of probiotic strains, e.g., belonging to the genera Lactobacillus, Bacillus, Pediococcus, and Weissella, which can be used in preparations for dietary supplements, food manufacturing, and pharmaceutical applications, with the aim of safely and rapidly degrading gluten into non-toxic, non-immunogenic digesta.

Description

[Technical field]

[0001] The present invention relates to a method for identifying a consortium of probiotic strains, e.g. belonging to the genera Lactobacillus, Bacillus, Pediococcus and Weissella, which can be used in preparations for dietary or pet food supplements, food or pet food production, and pharmaceutical applications, with the aim of safely and rapidly degrading gluten into non-toxic and non-immunogenic digesta. [Background technology]

[0002] Gluten is the major protein network in cereals such as wheat, rye and barley. It contains the monomeric α-, γ- and Ω-gliadins and possesses peptide sequences with immunogenic and / or toxic potential (the most prominent examples are shown in Table 1).

[0003] [Table 1]

[0004] Thus, dietary ingestion of gluten may cause health disorders if the digestion of gliadin or glutenin is incomplete and toxic peptides are released in sensitive individuals. The range of gluten-related disorders includes celiac disease (CD), wheat allergy (WA), non-celiac gluten sensitivity (NCGS), and gluten-sensitive irritable bowel syndrome [1]. Currently, there is no treatment available for these disorders, and avoiding gluten intake is the only effective solution, especially for those suffering from CD. Interestingly, gluten avoidance has been suggested to be effective for various other health conditions (e.g., schizophrenia, atopy, fibromyalgia, endometriosis, obesity, nonspecific gastrointestinal symptoms) [2]. These facts shed light on the rise of the gluten-free diet (GFD). And such practices are spreading to many healthy and symptom-free individuals. For example, it has been reported that 33% of the US population wants to avoid gluten, and 41% of the athletic population spends more than half of their time on a GFD [3]. However, implementing a GFD is not without challenges and side effects that must be weighed against the risks and benefits. People with CD are required to adhere strictly to a GFD, which can be difficult to achieve given that even foods considered or advertised as gluten-free often contain trace amounts of gluten above the safe intake limit of gluten (usually less than 20 ppm for CD patients). Ensuring food safety for patients with CD and related gluten-related disorders requires reliable and efficient strategies to support gluten avoidance or detoxification.

[0005] In the absence of clear indications to continue a GFD, i.e. when gluten avoidance is a lifestyle choice rather than a medical necessity, the harmful side effects of this diet must be considered. A GFD is often unbalanced, for example due to the calorie excess and increased sugar and saturated fat content found in many gluten-substitute foods, as well as the lack of micronutrients and dietary fiber due to the avoidance of grain products [4-6]. Potential harms of a GFD therefore include growth / developmental delays in children and adolescents, various malnutrition-related disorders, hyperlipidemia, hyperglycemia, and coronary artery disease [6]. Moreover, long-term adherence to a GFD can lead to dysbiosis of the gut microbiota, with subsequent adverse health consequences [7].

[0006] As has become evident from experiments with differentially colonized mice [8] and comparisons of microbiota between CD patients and healthy controls [9, 10], the gut microbiota is a key determinant of the intestinal fate of gluten and the physiological response to it. As a result, several approaches targeting the microbiota have been developed to search for treatment options for gluten-related disorders. These approaches can be categorized as follows: 1: Oral administration of Lactobacillus or Bifidobacterium to cure dysbiosis associated with GFD or gluten-related disorders. 2: Oral administration of Lactobacillus or Bifidobacterium as a non-specific support of gluten-related disorders via undetermined mechanisms. 3: Oral administration of Lactobacillus or Bifidobacterium to support gluten degradation. 4: Oral administration of hydrolases peptide hydrolases isolated from fungi or bacteria ("glutenases") to support gluten degradation. So far, all these approaches have failed to provide consistent benefits to those in need. Moreover, the application of peptide hydrolases has been discussed as a possible health risk, since it may cause incomplete digestion of gluten, resulting in the release of toxic epitopes, exacerbating and not reversing gluten toxicity

[11] . The effectiveness of enzyme therapy for CD patients is also limited by the low proteolytic resistance and the limited extent and duration of enzyme activity during gastrointestinal transit

[12] .

[0007] Recently, the bacterial classification of several species of the genus Lactobacillus has been updated according to Zheng J, Wittouck S, Salvetti E, Cmap Franz HMB, Harris P, Matarelli PW, O'Toole B, Pot P, Vandamme J, Walter K, Watanabe S, Wuyts GE, Felis MG, Ganzle A, and Lebeer S (2020). Taxonomic notes on the genus Lactobacillus: description of 23 new genera, revised description of the genus Lactobacillus Beijerinck 1901, and merging of the Lactobacillaceae and Leuconostochaetae families. International Journal of Systematic and Evolutionary Microbiology. https: / / doi.org / 10.1099 / ijsem.0.004107. Of particular relevance in the present context are the following species:

[0008] [Table A]

[0009] We believe that the lack of benefit from probiotic interventions is due to an inappropriate selection and mixing of probiotic strains. A meaningful and active selection process is a prerequisite for identifying a consortium of probiotic bacteria that promotes rapid and complete glutenin digestion and interacts synergistically. Such a process has not been previously described and is the subject of the present invention.

[0010] Rashmi et al. disclosed four gluten-hydrolyzing Bacillus strains that were resistant to pH 2 and bile acids

[13] . However, the digests were not evaluated for their putative immunogenicity and expression of immunogenic peptides. Similarly, the specific peptidase activities of the strains alone or in combination were not evaluated. The gluten hydrolyzing potential of the consortium was not evaluated.

[0011] isolated nine Bacillus strains from traditional Thai fermented foods. They were evaluated by western blotting of gliadin hydrolysis using crude bacterial extracts. Neither the digestion products nor the immunogenic potential of the digests were characterized

[14] .

[0012] Clark et al. isolated 50 bacterial strains from the pig ileum by selective culture and screened them for PepN, PepI and PEP activity (corresponding to part of step 4) (Non-Patent Document 1).

[0013] Similarly, Fernandez et al.

[15] obtained 150 isolates from human saliva using selective culture and evaluated the strains for hydrolysis of gliadin, tripeptides, and 33-mers.

[0014] Patent document 1 claims a method for selecting lactic acid bacteria strains for use in the treatment of celiac disease, comprising the steps of selecting strains capable of degrading the 33-mer, 20-mer peptide QQLPQPQQPQQSPFQQQRPF, the 13-mer peptide LGQQQPFPPQQPY and the 18-mer peptide PQLPYPQPQLPYPQPQPF, which strains are capable of degrading said peptides in the presence of lysozyme, pepsin, chymotrypsin and trypsin at pH values ​​between 4 and 6.

[0015] evaluated the peptidase activity of Lactobacillus strains in vitro. Strains of the species Lactobacillus plantarum (Lactiprantibacillus plantarum), Lactobacillus bulgaricus, Lactobacillus rhamnosus, Lactobacillus paracasei (Lacticaseibacillus paracasei) and Lactobacillus casei (Lacticaseibacillus casei) showed activities of up to 10 mU / mg for PepN, 10 mU / mg for PepI, 5 mU / mg for PEP and 25 mU / mg for PepQ. When ten of these strains were applied in combination, the gliadin epitopes listed in Table 1 were hydrolyzed after 24 hours of incubation. The survival of the strains under gastric and small intestinal conditions was not measured, so the effectiveness of these strains in terms of gluten digestion in the human gastrointestinal tract cannot be predicted.

[0016] Herran et al. isolated 27 bacterial strains from the human small intestine, belonging to the species L. salivarius, L. rhamnosus, L. reuteri (Lactobacillus reuteri), L. casei (Lacticaceibacillus casei), L. oris, L. gasseri, L. fermentum, L. crispatus, L. brevis (Lactobacillus brevis), Bacillus subtilis, B. amyloliquefaciens, B. pumilus, and B. licheniformis. They showed proteolytic activity against the 33-mer only after a very long incubation of 24 hours, but not against other peptides

[17] . Similarly, weak activity against this epitope was seen in the case of other bacterial strains from the human small intestine, again including B. subtilis, B. pumilus, and B. licheniformis

[18] . [Prior art documents] [Patent documents]

[0017] [Patent Document 1] U.S. Patent Publication No. 2013 / 0121976A1 [Non-patent literature]

[0018] [Non-Patent Document 1] Journal of Allergy and Clinical Immunology, (February 2011) Volume 127, No. 2, Supp. SUPPL. 1, pp. AB243, Abstract No: 942, Meeting Information: 2011 American Academy of Allergy, Asthma and Immunology, AAAAI Annual Meeting, San Francisco, CA, USA, March 18, 2011 - March 22, 2011, ISSN: 0091-6749 Summary of the Invention [Problem to be solved by the invention]

[0019] The present invention discloses a method for identifying a consortium of probiotic strains, e.g., belonging to the genera Lactobacillus, Bacillus, Pediococcus, and Weissella, which can be used for complete and rapid gluten degradation in food and pharmaceutical applications.

[0020] Libraries of bacterial strains derived from gluten-exposed ecological niches (e.g. soil, processed cereals, sourdough, feces, human / animal gastrointestinal specimens) are subjected to successive screening steps consisting of resistance to simulated gastrointestinal conditions, suitable protease activity against gluten, and suitable peptidase activity against synthetic proline-containing peptide substrates. Strains passing these screening steps are combined with consortia (of living cells or their extracts) with complementary peptidase activity and tested for hydrolysis of gluten-induced relevant immunogenic peptides. Consortia that promote rapid and complete removal of these peptides are then utilized in gluten hydrolysis experiments under simulated gastrointestinal conditions. Digesta are then examined for the absence of gluten, gluten-induced immunogenic peptides, and immunogenic potential in duodenal explants from celiac disease patients. (See Figure 1). Finally, the consortium will be tested in vivo by a gluten challenge test that includes assessment of fecal samples for gluten content, gluten-induced immunogenic peptides, and microbiota composition analysis, including the content of introduced strains.

[0021] The screening process disclosed below provides a funnel for generating a consortium of probiotic strains that can be used in food manufacturing (gluten-free foods), as well as dietary supplements and pharmaceutical applications (aiding in the safe clearance of gluten in the intestine).

[0022] The subject of the present invention is therefore a method for identifying a consortium of probiotic strains that can be used to enhance the digestion of gluten and gluten-derived peptides (epitopes), At a minimum, the following steps: 1) providing a library of at least 10 probiotic bacterial strains; 2) culturing the probiotic bacterial strains of step 1) for at least 30 minutes under simulated gastric conditions (pH 1-4) and for at least 30 minutes under simulated intestinal conditions (pH 5.5-8.5), and selecting strains that show a CFU reduction of less than 2 log after the simulated gastrointestinal conditions; 3) measuring the proteinase activity against gluten of the strains selected in step 2) and selecting a strain capable of reducing the initial gluten level, which is at least 5000 ppm, by 10 to 70%; 4) measuring the activities of peptidases, aminopeptidase N (PepN); PepI, PepO, prolyl endopeptidyl peptidase (PEP); PepX, and PepQ peptide hydrolases, of the strains selected in step 3), and selecting strains having a peptidase activity of at least 1 U / g for at least one of these peptidases; 5) combining at least two strains selected in step 4) into a consortium of probiotic strains having an activity of peptidases PepN, PepI, PepO, PepX and PepQ of at least 1 U / g for each peptidase; 6) measuring the peptidase activity of the consortium of step 5) against the 12-mer peptide QLQPFPQPQLPY (Seq-ID No: 1), the 14-mer peptide PQPQLPYPQPQSFP (Seq-ID No: 2), the 20-mer peptide QQLPQPQQPQQSFPQQQRPF (Seq-ID No: 3), and the 33-mer peptide LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (Seq-ID No: 4), and selecting the consortium having peptidase activity that degrades all four epitopes by more than 50%; 7) measuring the peptidase activity of the consortium selected in step 6) for hydrolyzing gluten with an initial concentration of at least 5000 ppm under simulated gastric conditions (pH 1-4) for at least 30 minutes and under simulated intestinal conditions (pH 5.5-8.5) for at least 30 minutes, and selecting consortiums that reduce the initial gluten level to at least 5000 ppm and reduce the concentration of hydrolyzed residual gluten to less than 200 ppm; The method includes:

[0023] It is preferable to use a larger number of probiotic strains and to screen a larger variety of strains, and therefore in an advantageous configuration the library provided comprises at least 20, preferably at least 30, more preferably at least 40 and most preferably at least 50 probiotic strains.

[0024] In step 6), it is preferable to select a strain that has peptidase activity that degrades all four epitopes by more than 70%, preferably more than 90%.

[0025] In a preferred embodiment, the enzymatic activity of the peptidases aminopeptidase type N (PepN); PepI, PepO, prolyl endopeptidyl peptidase (PEP); PepX, and PepQ peptide hydrolases is at least 3 U / g (PepP), 5 U / g (PepO), 20 U / g (PepX), 17 U / g (PepI), 20 U / g (PepN) for at least one of these peptidases.

[0026] The peptidase activity of step 7) may be measured, for example, by ELISA using an appropriate antibody directed against a Pro-rich peptide sequence.

[0027] In a preferred configuration, the method further comprises one or more of the following steps: 8) measuring the hydrolysis of gluten during digestion of wheat bread (1 to 100 g of wheat bread) by the mixture of strains selected in step 6) under simulated gastrointestinal conditions, and selecting a strain that can degrade the gluten content of the wheat bread to less than 20 ppm within 6 to 24 hours and does not contain gluten-derived epitopes (12-mer peptides, 14-mer peptides, 20-mer peptides, and 33-mer peptides) after 180 minutes of simulated intestinal digestion; 9) Measuring the immunogenicity of the mixture of strains selected in step 7) using small intestinal tissue explants from CD patients by measuring the expression of the cytokines interleukin 2 (IL-2), interleukin 10 (IL-10), and interferon gamma (IFN-γ) after 6 to 48 hours of culture under gastrointestinal conditions, and selecting strains whose immunogenicity is equal to or less than that of the negative control.

[0028] The gastric conditions in steps 1) and 7) may include culturing the strain in simulated gastric fluid containing pepsin (0.5-6 g / L) at a temperature of 35° C.-39° C. for 30 minutes-300 minutes at a pH of 1-4; The intestinal conditions in step 1) may include culturing the strain in simulated intestinal fluid containing pancreatin (0.02-0.6% w / v) and bile salts (0.05-0.6%) at a temperature of 35° C.-39° C. for 30-300 minutes at a pH of 5.5-8.5.

[0029] In step 4), the activities of the peptidases aminopeptidase N-type (PepN); PepI, PepO, prolyl endopeptidyl peptidase (PEP); PepX, and PepQ peptide hydrolases may be measured using a strain in the form of live cells or a cytoplasmic extract thereof, with a strain density of 7.0 to 11.0 log CUF / mL and a peptide substrate having an amino acid sequence suitable for detecting the activities of aminopeptidase N-type (PepN); PepI, PepO, prolyl endopeptidyl peptidase (PEP); PepX, and PepQ peptide hydrolases.

[0030] The peptidase activity in step 6 may be measured using live cells or their cytoplasmic extracts in a buffered medium (pH 6.0-9.0) at 35°C-39°C for 1-12 hours, and a strain capable of degrading all four epitopes by more than 95%, preferably more than 98%, is selected.

[0031] Step 8) simulated gastrointestinal conditions include: - the strains selected in step 7) with a strain density between 7.0 and 11.0 log CUF / mL; - their cytoplasm, and / or - Bacillus protease Incubation in simulated gastric fluid containing pepsin (0.5-6 g / L) at a temperature of 36.5°C to 37°C for 30-300 minutes at a pH of 2-4; - the strain selected in step 7), - its cytoplasm, and / or - Bacillus protease Incubation in simulated intestinal fluid containing pancreatin (0.02-0.6% w / v) and bile salts (0.05-0.6%) at temperatures between 36.5°C and 37°C for 30 minutes to 48 hours at a pH of 7.0-8.5. may be included.

[0032] In a preferred configuration, the bacterial strain is derived from one or more of soil; cereals (wheat, rye, barley); cereal products; sourdough; human, pig, dog, cat, rat or mouse feces; human, pig, dog, cat, rat or mouse gastrointestinal tract specimens.

[0033] In an advantageous configuration, the bacterial strain is selected from one or more of the genera Lactobacillus, Bacillus, Pediococcus and Weissella.

[0034] The method according to the invention results in a consortium of probiotic strains that provides a technical solution for gluten digestion, based on the following considerations: - The breakdown of gluten / gliadin / glutenin during human digestion is not beneficial in itself, as incomplete breakdown can lead to the generation of toxic and / or immunogenic peptides. - Concerns have been expressed about the safety of currently available means of inducing gluten degradation in vivo, as these may only partially degrade gluten and therefore may induce or exacerbate gluten toxicity. - If we are to induce gluten degradation in vivo, we need to ensure that such degradation is complete and that safe degradation products result. - Given the diversity of immunogenic gluten-specific peptide sequences, a combination of peptide hydrolases from different microorganisms is required to ensure complete degradation of all peptides. - such a combination is preferably provided by a consortium of probiotic microorganisms, i.e. a consortium of probiotic microorganisms which are metabolically active and which act synergistically with each other in the relevant parts of the gastrointestinal tract (i.e. the stomach and the duodenum) to promote the safe, rapid and complete digestion of the gluten proteins of the relevant food matrix into non-toxic, non-immunogenic small peptides or amino acids. - We believe that such synergistic effects can be achieved by combining acid- and bile-tolerant bacterial strains with appropriate protein / peptide substrate specificity, and have found that combinations of specific Lactobacillus and Bacillus species (including their cytoplasmic extracts) from specific ecological niches are particularly useful.

[0035] The probiotic consortium selected according to our method could benefit people in need in the following ways: (i) The safe elimination of intentionally or accidentally ingested gluten as a treatment or complementary therapy for patients with CD, WA and NCGS, with the possibility of reverting to a conventional gluten-containing diet. (ii) The safe elimination of intentionally or accidentally ingested gluten as a treatment or complementary therapy for people with non-specific intestinal or extra-intestinal symptoms that can be attributed to gluten ingestion, with the possibility of returning to a conventional gluten-containing diet. (iii) Providing a solution for asymptomatic people wishing to minimize their gluten exposure as an alternative to complying with the GFD. [Brief description of the drawings]

[0036] [Figure 1A]Figure 1A shows the score plot of the first and second principal components after principal component analysis (PCA) based on the common aminopeptidase N-type (PepN), proline iminopeptidase (PepI), X-prolyl dipeptidyl aminopeptidase (PepX), endopeptidase (PepO), and prolyl endopeptidase (PepP) activities of cytoplasmic extracts of 119 Bacillus, Lactobacillus, Pediococcus, and Weissella strains. PepN, PepI, PepX, and PepP were measured using Leu-p-nitroanilide (p-NA), Pro-p-NA, Gly-Pro-p-NA, Z-Gly-Gly-Leu-p-NA, and Z-Gly-Pro-4-nitroanilide substrates, respectively. Strains showing very high peptidase activity (for at least one peptidase) show more than two factor 2s and at least one factor 1. [Figure 1B] Figure 1B shows the loading plots of the first and second principal components after principal component analysis (PCA) based on the common aminopeptidase N-type (PepN), proline iminopeptidase (PepI), X-prolyl dipeptidyl aminopeptidase (PepX), endopeptidase (PepO), and prolyl endopeptidase (PepP) activities of cytoplasmic extracts of 119 Bacillus, Lactobacillus, Pediococcus, and Weissella strains. PepN, PepI, PepX, and PepP were measured using Leu-p-nitroanilide (p-NA), Pro-p-NA, Gly-Pro-p-NA, Z-Gly-Gly-Leu-p-NA, and Z-Gly-Pro-4-nitroanilide substrates, respectively. [Diagram 2] Figure 2 shows the peptidase activities (PepN, PepI, PepX, PepO, and PepP) of selected single Bacillus (B.), Lactobacillus (L.), and Pediococcus (P.) strains. One unit (U) of activity was defined as the amount of enzyme required to liberate 1 μmol of p-nitroaniline per minute under the assay conditions. [Diagram 3] FIG. 3 shows the peptidase activity of the strain mixture against immunogenic epitopes. [Figure 4A]FIG. 4A shows the RP-HPLC peptide profile of the control digested wheat bread sample. [Figure 4B] FIG. 4B shows the RP-HPLC peptide profile of a wheat bread sample digested with Mixture 4. [Figure 4C] FIG. 4C shows the RP-HPLC peptide profile of a wheat bread sample digested with Mixture 7. [Figure 5A] Figure 5A shows the concentration of interleukin 2 (IL-2) (ng / μL) in duodenal biopsy specimens from CD patients. Control: wheat bread digested without the addition of bacterial cells and microbial enzymes; RPMI + gastric and intestinal fluids: negative control; microbial consortium 4: wheat bread digested with live and lysed cells of L. plantarum (Lactiplantibacillus plantarum) DSM 33363 and DSM 33364, L. paracasei (Lacticaseibacillus paracasei) DSM 33373, Bacillus subtilis DSM 33298 and Bacillus pumilus DSM 33301, and with the addition of E1, E2, VeronPS, VeronHPP commercial enzymes; microbial consortium 7: L. plantarum (Lactiplantibacillus plantarum) DSM 33362, DSM 33366 and DSM 33370, L. reuteri (Rimocilla Wheat bread digested with live and lysed cells of Lactobacillus reuteri DSM 33374, Bacillus megaterium DSM 33356, and Bacillus subtilis DSM 33353, plus E1, E2, VeronPS, and VeronHPP commercial enzymes; and wheat bread digested with live and lysed cells of microbial consortium 16: L. plantarum (Lactiplantibacillus plantarum) DSM 33363, DSM 33364, L. paracasei (LacticaseiBacillus paracasei) DSM 33373, L. reuteri (Lactobacillus reuteri) DSM 33374, Bacillus megaterium DSM 33330, Bacillus pumilus DSM 33297, DSM 33355. CD1–CD10 are duodenal biopsy specimens from patients with celiac disease. [Figure 5B]Figure 5B shows the concentration (ng / µL) of interleukin 10 (IL-10) in duodenal biopsy specimens from CD patients. Samples and microbial consortia are the same as in Figure 5A. [Figure 5C] Figure 5C shows the concentration (ng / µL) of interferon gamma (IFN-γ) in duodenal biopsy specimens from CD patients. Samples and microbial consortia are the same as in Figure 5A. [Figure 6] FIG. 6 is a schematic diagram of the process of the present invention for identifying a consortium of probiotic microorganisms from the genera Lactobacillus, Bacillus, Pediococcus and Weissella for use in the safe, rapid and complete degradation of gluten in humans and animals. EXAMPLES

[0037] Process step description An overview of the process is shown in Figure 6. Step 1: Accumulation of a library of bacterial strains The following libraries contain bacterial strains that may be suitable as starting points for the present invention. For example, we have accumulated four strain libraries: Lactobacillus (library 1), Bacillus (library 2), Pediococcus (library 3), and Weissella (library 4). Each library contains at least 10 different strains. The strains are derived from soil; cereals (wheat, rye, barley); cereal products; sourdough; human, pig, dog, cat, rat or mouse feces; and human, pig, dog, cat, rat or mouse gastrointestinal specimens. The strains belong to the following genera:

[0038] Library 1 = Lactobacillus Strains include, for example, Lactobacillus plantarum (Lactiprantibacillus plantarum), Lactobacillus paracasei (Lacticaseibacillus paracasei), Lactobacillus sanfrancisensis (Fructilactobacillus sanfrancisensis), Lactobacillus brevis (Reviractobacillus brevis), Lactobacillus casei (Lacticaseibacillus casei), Lactobacillus rossiae, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus spp. The lactobacillus species may belong to the following genus: Lactobacillus, Lactobacillus crispatus, Lactobacillus carbatus, Lactobacillus delbrueckii, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus johnsonii, Lactobacillus kefiri, Lactobacillus mucosae, Lactobacillus reuteri (rimosyllactobacillus reuteri), Lactobacillus rhamnosus, Lactobacillus sakei, or Lactobacillus salivarius. Preferably, the strain belongs to the species Lactobacillus plantarum (Lactiplantibacillus plantarum), Lactobacillus paracasei (Lacticaseibacillus paracasei), Lactobacillus sanfrancisensis (Fructylactobacillus sanfrancisensis), Lactobacillus brevis (Reviractobacillus brevis) or Lactobacillus casei (Lacticaseibacillus casei).

[0039] Library 2 = Bacillus The strain may, for example, belong to the Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Bacillus amyloliquefaciens group, Bacillus coagulans, Bacillus fusiformis, or Bacillus megaterium. Preferably, the strain belongs to the Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, or Bacillus megaterium species.

[0040] Library 3 = Pediococcus The strain may belong, for example, to Pediococcus acidilactici, Pediococcus dextrinicus, Pediococcus parvulus, or Pediococcus pentosaceus.

[0041] Library 4 = Weissella The strain may, for example, belong to Weissella confusa, Weissella cibaria, Weissella halotolerans, Weissella kandleri, or Weissella paramesenteroides.

[0042] Step 2: Resistance of strains to simulated gastric and intestinal conditions Simulated gastric and intestinal fluids were prepared and used as described by Fernandez et al.

[19] . Stationary-phase growing cells were harvested at 8,000 g for 10 min, washed with physiological solution, and suspended in 50 mL of simulated gastric fluid (cell density: 10 log CFU / mL). The simulated solution contained NaCl (125 mM / L), KCl (7 mM / L), NaHCO3 (45 mM / L), and pepsin (3 g / L)

[20] . The final pH was adjusted to 2.0, 3.0, and 8.0. The pH value of 8.0 was used to investigate the effects of the components of the simulated gastric fluid apart from the effects of low pH

[19] . The suspension was incubated at 37 °C under anaerobic conditions with stirring to artificially induce peristalsis. Aliquots of this suspension were taken at 0, 90, and 180 min to measure the viable cell count. The effect of gastric digestion was also measured by suspending cells in reconstituted skim milk (RSM) (11% solids, w / v) before inoculation with simulated gastric fluid at pH 2.0. The final pH after addition of RSM was approximately 3.0. This condition was analyzed to simulate the effect of the food matrix on gastric passage

[20] . After 180 min of gastric digestion, cells were harvested and suspended in simulated intestinal fluid containing 0.1% (w / v) pancreatin and 0.15% (w / v) oxgall bile salts (at pH 8.0). The suspension was incubated at 37 °C with stirring and aliquots were taken at 0, 90 and 180 min

[21] . Selection criteria = CFU loss less than 2 logs.

[0043] Step 3: Proteinase activity of the strains against gluten 24-h-old cells of bacterial strains were harvested by centrifugation (12,400 × g for 10 min at 4 °C), washed with sterile 0.05 M potassium phosphate buffer (pH 7.0), resuspended in the same buffer at an absorbance at 620 nm (A620): 2.5 (corresponding to a cell density of approximately 9.0 log CFU / mL) and used for the enzyme assay. Proteinase (cell envelope-bound proteinase) activity was measured using wheat flour proteins as substrates. Flour proteins were extracted from wheat flour according to the method of Weiss et al.

[22] . The assay mixtures containing 4 mg / mL albumin / globulin, gliadin, or glutenin in 0.05 M potassium phosphate buffer (pH 7.0) and 0.1 mL of cell suspension (approximately 9.0 log CFU / mL) were incubated at 37 °C for 180 min under stirring conditions (150 rpm). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 12.5% ​​acrylamide gels stained with B10 Bio-Safe Coomassie blue. A low range SDS-PAGE molecular weight standard was used. Three gels of each assay were analyzed for protein band intensity by the Quantity One software package. Gliadin (4 mg / mL) was suspended in gastric juice at pH 2.0 and incubated at 37°C for 180 min under stirring (150 rpm). After gastric digestion, the hydrolyzed gliadin was centrifuged at 10,000 rpm for 10 min, and then intestinal juice with (treated) or without (control) 0.1 mL of cell suspension (approximately 9.0 log CFU / mL) was added separately to the supernatant (containing soluble peptides) and the pellet (containing insoluble peptides and proteins). The assay mixture was incubated at 37°C for 180 min under stirring (150 rpm). Aliquots of the intestinal suspension were incubated for 30 h at 37°C under stirring conditions (150 rpm).

[0044] Protein concentrations were measured by the Bradford method

[23] . Peptide concentrations were measured by the o-phthaldialdehyde (OPA) method (Church FC, Swaisgood HE, Porter DH, Catignani GL, 1983. A spectrophotometric assay with o-phthaldialdehyde for measuring proteolysis of milk and isolated milk proteins. J. Dairy Sci, 66:1219-1227). A standard curve constructed with tryptone (0.25-1.5 mg / mL) was used as a reference. A similar standard curve was obtained using peptone. Immunological analysis was performed using the R5 antibody-based sandwich and competitive ELISA (R5-ELISA). The R5-ELISA by Valdes et al.

[24] was performed using the RIDASCREEN® Gliadin Competitive Detection Kit according to the manufacturer's instructions (R-Biopharm AG, Germany). Selection criteria: Very high gluten degradation compared to other strains.

[0045] Step 4: Peptidase activity of the strains on synthetic substrates Late logarithmic-phase cultures of each strain (approximately 9.0 log CFU / mL) were used to analyze cytoplasmic peptidase activity. An aliquot of the washed cell pellet (0.3 g [dry weight]) was resuspended in 50 mM Tris-HCl (pH 7.0), incubated at 30°C for 30 min, and centrifuged at 13,000 × g for 10 min to remove loosely bound enzymes on the cell surface. Cytoplasmic extracts were prepared by incubating the bacterial suspension with lysozyme in 50 mM Tris-HCl (pH 7.5) buffer containing 24% sucrose under stirring (approximately 160 rpm) for 60 min at 37°C. Spheroplasts were resuspended in isotonic buffer and sonicated at 16 A / s for 40 s (Sony Prep model 150; Sanyo, UK). Cytoplasmic extracts were concentrated 10-fold by lyophilization, resuspended in 5 mM Tris-HCl (pH 7.0), and dialyzed at 4°C for 24 h.

[0046] General aminopeptidase type N (PepN), proline iminopeptidase (PepI), and X-prolyl dipeptidyl aminopeptidase (PepX) activities of Lactobacillus cytoplasmic extracts were measured using Leu-p-nitroanilide (p-NA), Pro-p-NA, and Gly-Pro-p-NA substrates, respectively. The assay mixture contained 900 μL of 2.0 mM substrate in 0.05 M potassium phosphate buffer (pH 7.0) and 100 μL of cytoplasmic extract. The mixtures were incubated at 37°C for 180 min, and the absorbance was measured at 410 nm. The data were compared to a standard curve generated using p-nitroaniline. One unit of activity was defined as the amount of enzyme required to liberate 1 μmol of p-nitroaniline in 1 min under the assay conditions. Selection criteria = very high activity for at least one enzyme compared to other strains.

[0047] Step 6: Peptidase activity of a consortium of strains against Pro-rich synthetic gluten-derived epitopes A diverse mixture (consortium) of bacterial strains was used to analyze their ability to degrade in vitro immunogenic epitopes responsible for gluten intolerance. Fragment 57-68 of α9-gliadin (QLQPFPQPQLPY), fragment 62-75 of A-gliadin (PQPQLPYPQPQSFP), fragment 134-153 of γ-gliadin (QQLPQPQQPQQSFPQQQRPF), and fragment 57-89 of α2-gliadin (LQLQPFPQPQLPYPQPQLPYP-QPQLPYPQPQPF) (33-mer) were chemically synthesized. Hydrolysis of peptides was performed using cytoplasmic extracts of preselected bacterial strains. A mixture containing 100 μL of cytoplasmic extract and 2 mM synthetic peptide in 1 mL of 50 mM phosphate buffer (pH 7.5) was incubated with stirring (150 rpm) at 37 °C for 180 min. Peptide hydrolysis was monitored and individual HPLC analysis was used to look for free peptides. Analysis was completed using liquid chromatography coupled with electrospray ionization (ESI)-ion trap mass spectrometry (MS). Selection criteria: Degradation of all four epitopes

[0048] Step 7: Hydrolysis of gluten by a consortium of strains under simulated gastrointestinal conditions Commercially available gliadin was used to test its ability to hydrolyze gluten, in relation to the procedure described by Francavilla et al.

[16] . Gliadin (4 mg / mL) was suspended in simulated gastric fluid containing NaCl (125 mM / L), KCl (7 mM / L), NaHCO3 (45 mM / L), and pepsin (3 g / L). The final pH was adjusted to 2.0 with HCl. The suspension was incubated at 37 °C under anaerobic conditions with stirring to induce artificial peristalsis. After 180 min of gastric digestion, the hydrolyzed gliadin was centrifuged at 10,000 rpm for 10 min, and the supernatant (containing soluble peptides) and pellet (containing insoluble peptides and proteins) were added separately to the simulated intestinal fluid. The simulated intestinal fluid contained 0.1% (w / v) pancreatin and 0.15% (w / v) oxgol bile salts (Sigma-Aldrich Co.) at pH 8. Simulated intestinal fluid with (treated) or without (control) 0.1 mL of cell suspension (approximately 9.0 log CFU / mL) was incubated at 37°C under stirring (150 rpm) for 180 min. Protein concentration was measured by the Bradford method

[23] . Peptide concentration was measured by the o-phthaldialdehyde (OPA) method (Church FC, Swaisgood HE, Porter DH, Catignani GL, 1983; A spectrophotometric assay using o-phthaldialdehyde for measuring proteolysis of milk and isolated milk proteins. J. Dairy Sci. 66:1219-1227). A standard curve constructed using tryptone (0.25–1.5 mg / mL) was used as a reference. A similar standard curve was obtained using peptone. Immunological analysis was performed using the R5 antibody-based sandwich and competitive ELISA (R5-ELISA). R5-ELISA

[24] was performed using the RIDASCREEN® Gliadin Competitive Detection Kit according to the manufacturer's instructions (R-Biopharm AG, Germany). Selection criteria: Associated hydrolysis of gluten during incubation.

[0049] Step 9: Evaluating the safety of gluten hydrolysis by a consortium of strains using small intestinal tissue explants from CD patients Duodenal biopsy specimens were obtained from 10 patients with CD (age range: 19–30 years) after GFD. All CD patients expressed the HLA-DQ2 phenotype. CD was diagnosed according to the criteria of the European Society of Paediatric Gastroenterology, Hepatology and Nutrition (European Society of Paediatric Gastroenterology and Nutrition, 1990, Revised Criteria for the Diagnosis of Celiac Diseases, Report of the Working Group of the European Society of Paediatric Gastroenterology and Nutrition, Arch Dis Child 65:909–911). All biopsy specimens were placed in ice-cold medium (RPMI 1640; Gibco-Invitrogen, UK) immediately after resection and transported to the laboratory within 30 min. Duodenal biopsy specimens were cultured for 4 h using the organ tissue culture method originally described by Browning and Trier

[25] . Briefly, the biopsy specimens were placed villous side up on a stainless steel mesh and placed on the central well of an organ tissue culture dish (Falcon, USA). Wells contained RPMI supplemented with 15% fetal bovine serum (Gibco-Invitrogen) and 1% penicillin-streptomycin. Dishes were placed in anaerobic jars and incubated at 37°C.

[0050] Four biopsy specimens from each CD patient were cultured in media under four conditions: (i) a dough containing a mixture of bacterial strains and an enzyme mixture (E1, E2, VeronPS, VeronHPP) digested for 48 h; (ii) a dough containing another mixture of bacterial strains and an enzyme mixture (E1, E2, VeronPS, VeronHPP) digested for 48 h; (iii) a control dough digested for 48 h (control); (iv) medium (RPMI1640 + gastric and intestinal fluids, negative control).

[0051] Biopsy specimens from each patient were rinsed and stored in RNAlater at -80°C to preserve the RNA. Total RNA was extracted from tissues using the RNeasy mini kit (Qiagen GmbH) according to the manufacturer's instructions. The concentration of mRNA was estimated by measuring UV absorbance at 260 nm. An aliquot of total RNA (500 ng) was reverse transcribed using random hexamers, TaqMan reverse transcription reagents, and 3.125 U / μL of MultiScribe reverse transcriptase to a final volume of 50 μL. cDNA samples were stored at -20°C.

[0052] RT-PCR of IFN-γ, IL-2, and IL-10 genes: RT-PCR was performed in 96-well plates using an ABI Prism 7500HT rapid sequence detection system (Applied Biosystems). Data collection and analysis were performed using the machine software. PCR primers and fluorogenic probes for the target genes (IFN-γ, IL-2, and IL-10) and endogenous control (the gene encoding glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) were purchased as TaqMan gene expression assays and pre-developed TaqMan assays, respectively. The assays were procured as a 20x mixture of PCR primers and TaqMan minor groove binder 6-carboxyfluorescein dye-labeled probes with a non-fluorescent quencher at the 3' end. Two-step reverse transcription PCR was performed using first-strand cDNA with a final concentration of 1x TaqMan gene expression assay mix and 1x TaqMan universal PCR master mix. The final reaction volume was 25 μL. Each sample was analyzed in triplicate, and all experiments were repeated twice. A no-template control (RNase-free water) was included in every plate. The following thermal cycler conditions were used: 50 °C for 2 min (uracil DNA glycosylase activation); 95 °C for 10 min; 40 cycles of 95 °C for 15 s and 60 °C for 1 min. First, standard curve and validation experiments were performed for each primer / probe set. Six serial dilutions (20–0.1 ng / μL) of IFN-γ, IL-2, or IL-10 cDNA were used as templates for each primer / probe set. Standard curves were generated by plotting threshold cycle (CT) values ​​against the logarithm of the amount of input cDNA. The CT value is the PCR cycle at which an increase in the fluorescent reporter above baseline levels is first detected. The mean values ​​of the target genes were normalized using an endogenous reference gene (GAPDH gene). Healthy duodenal biopsy specimens were used to calibrate all experiments.Levels of IFN-γ, IL-2, and IL-10 proteins secreted into the supernatants were quantified by ELISA in 96-well round-bottom plates (Tema Ricerca, Milan, Italy) according to the manufacturer's recommendations. Selection criteria = non-immunogenicity of digests.

[0053] Example 1: Probiotic microorganisms resistant to gastrointestinal conditions Simulated gastric and intestinal fluids were used as described by Fernandez et al.

[19] . Stationary-phase growing cells were harvested at 8,000 g for 10 min, washed with physiological solution, and suspended in 50 mL of simulated gastric fluid (cell density: 10 log CFU / mL). The simulated gastric fluid contained NaCl (125 mM / L), KCl (7 mM / L), NaHCO3 (45 mM / L), and pepsin (3 g / L) (Sigma-Aldrich CO., St. Louis, MO, USA)

[20] . The final pH was adjusted to 2.0, 3.0, and 8.0. The pH 8.0 value was used to study the effects of the components of the simulated gastric fluid, apart from the effects of low pH. The suspension was incubated at 37 °C under anaerobic conditions and with stirring, to artificially induce peristalsis. Aliquots of this suspension were taken at 0, 90, and 180 min to measure the viable bacterial count. The effect of gastric digestion was also measured by suspending cells in reconstituted skim milk (RSM) (11% solids, w / v) before inoculation with simulated gastric fluid at pH 2.0. The final pH after addition of RSM was approximately 3.0. This condition was analyzed to simulate the effect of the food matrix on gastric passage

[20] . After 180 min of gastric digestion, cells were harvested and suspended in simulated intestinal fluid at pH 8.0 containing 0.1% (w / v) pancreatin and 0.15% (w / v) oxgall bile salts (Sigma-Aldrich Co.). The suspension was incubated at 37 °C with stirring and aliquots were taken at 0, 90, and 180 min

[21] . 119 of fewer than 400 strains tested had a population density of less than the initial 1 × 10 10 Those that showed less than a 2 log reduction in CFU / mL were defined as resistant to simulated gastrointestinal conditions.

[0054] Example 2: Protease and peptidase activity of a single strain resistant to gastrointestinal conditions All 119 strains (63 Lactobacillus spp.; 3 Weiss spp.; 1 Pediococcus spp.; and 51 Bacillus spp.) that showed resistance to simulated gastrointestinal conditions were tested for peptidase and proteinase activity against synthetic substrates. Late logarithmic growth phase cultures (approximately 9.0 log CFU / mL) of each strain were used to analyze peptidase activity. An aliquot (0.3 g [dry weight]) of the washed cell pellet was resuspended in 50 mM Tris-HCl (pH 7.0), cultured at 30 °C for 30 minutes, centrifuged at 13,000×g for 10 minutes to remove the enzyme loosely bound to the cell wall. The bacterial suspension was cultured with lysozyme in a buffer of 50 mM Tris-HCl (pH 7.5) containing 24% sucrose under stirring conditions (about 160 rpm) at 37 °C for 60 minutes to prepare a cytoplasmic extract. The spheroplasts were resuspended in an isotonic buffer and sonicated at 16 A / s for 40 seconds (Sony Prep model 150; Sanyo, UK). The extract was concentrated 10-fold by lyophilization, resuspended in 5 mM Tris-HCl (pH 7.0), and dialyzed at 4 °C for 24 hours. The activities of general aminopeptidase N type (PepN), proline iminopeptidase (PepI), X-prolyl dipeptidyl aminopeptidase (PepX), endopeptidase (PepO), and prolyl endopeptidase (PepP) in the cytoplasmic extract of Lactobacillus were measured using Leu-p-nitroanilide (p-NA), Pro-p-NA, Gly-Pro-p-NA, Z-Gly-Gly-Leu-p-NA, and Z-Gly-Pro-4-nitroanilide substrates (Sigma Chemical Co), respectively. The assay mixture contained 900 μL of 2.0 mM substrate in 0.05 M potassium phosphate buffer at pH 7.0 and 100 μL of the cytoplasmic extract. The mixture was cultured at 37 °C for 180 minutes, and the absorbance was measured at 410 nm. The data were compared with a standard curve prepared using p-nitroaniline. One unit of activity was defined as the amount of enzyme required to liberate 1 μmol of p-nitroaniline per minute under the assay conditions. Based on the principal component analysis (PCA) data from the above peptidase activities, several strains were clearly distinguished from other strains (Figure 1). Figure 2 reports strains showing very high peptidase activity (for at least one peptidase activity). The PepN activity ranged from 0.0 (U002-C04; U541-C05; U776-C02; DSM33301; U021-C01; DSM32540; U567-C04) to 31.400 ± 0.09 U (DSM33362) (median 3.08).Strains with low peptidase activity (with internal numbers and / or deposited at DSMZ: U002-C04; U541-C05; U776-C02; DSM33301; U021-C01; DSM32540; U567-C04) were not evaluated further. Other strains with the highest activity were DSM33367, DSM33374, DSM33370, DSM33371, DSM33377, DSM33373, Bacillus pumilus DSM33297, Bacillus subtilis DSM33298, DSM33376, DSM33375, DSM33363, Bacillus licheniformis DSM33354, and Bacillus megaterium DSM33356 (Figures 1 and 2). The median value for PepI was 1.66. The most active strains (PepI activity >18 U) were DSM33375 and DSM33373. PepX activity ranged from 0.0 to about 24 U. The most active strains were DSM33379, DSM33371, DSM33370, DSM33369, DSM33374, DSM33373, and DSM33363 (Figures 1 and 2) (median 1.81). The median value for PepO was 0.54. The most active strains (PepO activity >5 U) were DSM33353, DSM33355, and DSM33301. PepP activity ranged from 0.0 to 6.23 U (DSM33368) (median 0.22). Other most active strains (PepP activity >3 U) were Bacillus megaterium DSM33300, DSM33378, DSM33371, DSM33377, DSM33367, DSM33374, DSM33366, DSM33373 and DSM33364.

[0055] Figure 1 shows the scores (A) and loadings (B) plots of the first and second principal components after principal component analysis (PCA) based on the common aminopeptidase N-type (PepN), proline iminopeptidase (PepI), X-prolyl dipeptidyl aminopeptidase (PepX), endopeptidase (PepO), and prolyl endopeptidase (PepP) activities of cytoplasmic extracts of 119 Bacillus, Lactobacillus, Pediococcus, and Weissella strains. PepN, PepI, PepX, and PepP were measured using Leu-p-nitroanilide (p-NA), Pro-p-NA, Gly-Pro-p-NA, Z-Gly-Gly-Leu-p-NA, and Z-Gly-Pro-4-nitroanilide substrates, respectively. Strains showing very high peptidase activity (for at least one peptidase) are reported in red.

[0056] Figure 2 shows the peptidase activities (PepN, PepI, PepX, PepO and PepP) of selected single Bacillus (B.), Lactobacillus (L.) and Pediococcus (P.) strains. One unit of activity was defined as the amount of enzyme required to liberate 1 μmol of p-nitroaniline in 1 min under the assay conditions.

[0057] Experimental Example 3: Peptidase activity of strain mixtures against immunogenic epitopes Bacillus, Lactobacillus, and Pediococcus strains exhibiting very high peptidase activity (for at least one peptidase) were evaluated as a mixture of strains combining strong and complementary enzymatic activities. The different mixtures were used to analyze their ability to degrade in vitro immunogenic epitopes responsible for gluten intolerance.

[0058] Peptide hydrolysis was performed using a combination of cytoplasmic extracts of preselected bacterial strains. Immunogenic epitopes corresponding to fragment 57-68 of α9-gliadin (QLQPFPQPQLPY), fragment 62-75 of A-gliadin (PQPQLPYPQPQSFP), fragment 134-153 of γ-gliadin (QQLPQPQQPQQSFPQQQRPF), and fragment 57-89 of α2-gliadin (LQLQPFPQPQLPYPQPQLPYP-QPQLPYPQPQPF) (33-mer) were chemically synthesized and used at an initial concentration of 1 mM. Hydrolysis was monitored by RP-HPLC. Single peaks from RP-HPLC were analyzed by nano-ESI tandem mass spectrometry (nano-ESI-MS / MS). The strain mixtures that showed the best hydrolysis of the synthetic immunogenic epitopes were numbers 3, 4 and 5 (Figure 3), which completely hydrolyzed all toxic peptides (more than 90% hydrolysis). Figure 3 shows the peptidase activity of the strain mixtures against the immunogenic epitopes.

[0059] The strain mixture was as follows: 1. L. plantarum (LactiplanciBacillus plantarum) DSM33362, DSM33363, DSM33364, DSM33366; L. sanfrancisensis (Fructilactobacillus sanfrancisensis) DSM33379; Bacillus pumilus DSM33297, DSM33355, Bacillus licheniformis DSM33354, Bacillus megaterium DSM33300, Bacillus subtilis DSM33353. 2. L. paracasei (Lacticaseibacillus paracasei) DSM 33375, DSM 33376; L. plantarum (Lactiplantibacillus plantarum) DSM 33369, DSM 33368; L. sanfrancisensis (Fructilactobacillus sanfrancisensis) DSM 33378; Bacillus licheniformis DSM 33354, Bacillus megaterium DSM 33300, DSM 33356, Bacillus pumilus DSM 33297, DSM 33301. 3. L. plantarum (Lactiplantibacillus plantarum) DSM 33370, DSM 33363, DSM 33364, Lactobacillus paracasei (Lacticaseibacillus paracasei) DSM 33373, L. brevis (Levilactobacillus brevis) DSM 33377; Bacillus pumilus DSM 33297, DSM 33355, Bacillus licheniformis DSM 33354, Bacillus megaterium DSM 33300, Bacillus subtilis DSM 33353. 4. L. plantarum (Lactiplantibacillus plantarum) DSM 33362, DSM 33367, DSM 33368; L. paracasei (Lacticaseibacillus paracasei) DSM 33375; L. sanfrancisensis (Fructilactobacillus sanfrancisensis) DSM 33379; Bacillus pumilus DSM 33301, Bacillus megaterium DSM 33300, DSM 33356, Bacillus subtilis DSM 33298, DSM 33353. 5. L. plantarum (Lactiplantibacillus plantarum) DSM 3366, DSM 33369, Lactobacillus reuteri (Limocylactobacillus reuteri) DSM 33374; L. paracasei (Lacticaseibacillus paracasei) DSM 33376; Pediococcus pentosaceus DSM 33371, L. sanfrancisensis (Fructilactobacillus sanfrancisensis) DSM 33378; Bacillus licheniformis DSM 33354, Bacillus pumilus DSM 33301, Bacillus megaterium DSM 33300, DSM 33356, Bacillus subtilis DSM 33298. 6. L. plantarum (Lactiplantibacillus plantarum) DSM 33370, DSM 33367, Lactobacillus reuteri (Limocylactobacillus reuteri) DSM 33374; L. brevis (Levilactobacillus brevis) DSM 33377; Bacillus pumilus DSM 33301, Bacillus megaterium DSM 33300, DSM 33356, Bacillus subtilis DSM 33298.

[0060] Experimental example 4: Gluten degradation under simulated gastrointestinal conditions by different consortia Gluten degradation was evaluated under simulated gastrointestinal digestion. With the intention of developing a feasible technical solution for complete gluten degradation in vivo, we sought a minimal combination that included as few strains as possible and as many as necessary.

[0061] Using mixtures 1 to 6 from Example 3 as a starting point, a total of 22 strains (Lactobacillus plantarum (Lactiplantibacillus plantarum) DSM 33370, DSM 33362, DSM 33363, DSM 33364, DSM 33366, DSM 33368, DSM 33369 and DSM 33367; Lactobacillus reuteri (Limosilactobacillus reuteri) DSM 33374; Lactobacillus paracasei (Lacticaseibacillus paracasei) DSM 33376, Lactobacillus paracasei (Lacticaseibacillus paracasei) DSM 33373, DSM 33375; Lactobacillus brevis (Reviractobacillus The following consortia were prepared: Bacillus brevis (DSM 33377; Pediococcus pentosaceus DSM 33371; Bacillus pumilus DSM 33297, DSM 33355, DSM 33301, DSM 33355; Bacillus licheniformis DSM 33354; Bacillus megaterium DSM 33300, DSM 33356; and Bacillus subtilis DSM 33298, DSM 33353).

[0062] 1. L. plantarum (Lactiplantibacillus plantarum) DSM 33370, DSM 33363 and DSM 33364, L. paracasei (Lacticaseibacillus paracasei) DSM 33373, L. brevis (Reviractobacillus brevis) DSM 33377, Bacillus pumilus DSM 33297, DSM 33355, DSM 33301; 2. L. plantarum (Lactiplantibacillus plantarum) DSM 33362 and DSM 33367, DSM 33368, L. paracasei (Lacticaseibacillus paracasei) DSM 33375, Bacillus subtilis DSM 33298, Bacillus licheniformis DSM 33354, and Bacillus megaterium DSM 33300; 3. L. plantarum (Lactiplantibacillus plantarum) DSM 33366, DSM 33369, Lactobacillus reuteri (Limocylactobacillus reuteri) DSM 33374, L. paracasei (Lacticaseibacillus paracasei) DSM 33376, Pediococcus pentosaceus DSM 33371, Bacillus megaterium DSM 33356, and Bacillus subtilis DSM 33353; 4. L. plantarum (Lactiplantibacillus plantarum) DSM 33363 and DSM 33364, L. paracasei (Lacticaseibacillus paracasei) DSM 33373, Bacillus subtilis DSM 33298 and Bacillus pumilus DSM 33301; 5. L. brevis (Reviractobacillus brevis) DSM 33377, Pediococcus pentosaceus DSM 33371, L. plantarum (Lactiplantibacillus plantarum) DSM 33369, Bacillus pumilus DSM 33297 and Bacillus megaterium DSM 33300; 6. L. paracasei (LacticaseiBacillus paracasei) DSM 33375, L. plantarum (Lactiplantibacillus plantarum) DSM 33367, DSM 33368; Bacillus pumilus DSM 33355, and Bacillus licheniformis DSM 33354; 7. L. plantarum (Lactiplantibacillus plantarum) DSM 33370, DSM 33362 and DSM 33366, Lactobacillus reuteri (Limocilactobacillus reuteri) DSM 33374, Bacillus megaterium DSM 33356, Bacillus subtilis DSM 33353; 8. L. plantarum (Lactiplantibacillus plantarum) DSM 33363, DSM 33364, L. paracasei (Lacticaseibacillus paracasei) DSM 33375, L. reuteri (Limocilactobacillus reuteri) DSM 33374, B. megaterium DSM 33300, B. pumilus DSM 33297; 9. L. paracasei (Lacticaseibacillus paracasei) DSM 33375, L. plantarum (Lactiplantibacillus plantarum) DSM 33367, L. reuteri (Limocylactobacillus reuteri) DSM 33374, B. megaterium DSM 33300, B. pumilus DSM 33297, B. licheniformis DSM 33354; 10. L. plantarum (Lactiplantibacillus plantarum) DSM 33363, DSM 33364, DSM 33370, L. brevis (Levilactobacillus brevis) DSM 33377, B. pumilus DSM 33297, Bacillus megaterium DSM 33356; 11. L. plantarum (Lactiplantibacillus plantarum) DSM 33362, DSM 33367, DSM 33368, L. paracasei (Lacticaseibacillus paracasei) DSM 33375, B. megaterium DSM 33300, Bacillus subtilis DSM 33353; 12. L. plantarum (Lactiplantibacillus plantarum) DSM 33366, DSM 33369, L. reuteri (Limocylactobacillus reuteri) DSM 33374, L. paracasei (Lacticaseibacillus paracasei) DSM 33376, P. pentosaceus DSM 33371, B. pumilus DSM 33297, DSM 33355; 13. L. brevis (Reviractobacillus brevis) DSM 33377, P. pentosaceus DSM 33371, L. sanfrancisensis (Fructylactobacillus sanfrancisensis) DSM 33379, B. megaterium DSM 33300, B. pumilus DSM 33297; 14. L. plantarum (Lactiplantibacillus plantarum) DSM 33368, L. paracasei (Lacticaseibacillus paracasei) DSM 33375, L. sanfrancisensis (Fructylactobacillus sanfrancisensis) DSM 33378, B. megaterium DSM 33300, B. pumilus DSM 33297, B. licheniformis DSM 33354; 15. L. plantarum (Lactiplantibacillus plantarum) DSM 33362, DSM 33366, DSM 33370, L. reuteri (Rimosyllactobacillus reuteri) DSM 33374, L. sanfrancisensis (Fructylactobacillus sanfrancisensis) DSM 33378, DSM 33379, B. licheniformis DSM 33354, Bacillus subtilis DSM 33353; 16. L. plantarum (Lactiplantibacillus plantarum) DSM 33363, DSM 33364, L. paracasei (Lacticaseibacillus paracasei) DSM 33373, L. reuteri (Limocylactobacillus reuteri) DSM 33374, B. megaterium DSM 33300, B. pumilus DSM 33297, DSM 33355.

[0063] Five grams of wheat bread (chewed for 30 seconds and collected in a beaker containing 10 mL of 0.05 M NaK-phosphate at pH 6.0) or related dough was suspended in simulated gastric fluid containing NaCl (125 mM), KCl (7 mM), NaHCO3 (45 mM), and pepsin (3 g / L) (Sigma-Aldrich CO., St. Louis, MO, USA). The suspension was added to the stock of selected strains as live (final cell density: approximately 9.0 log CFU / mL) lysed bacteria (corresponding to 9.0 log cells / mL). The calculated initial amount of gluten in the reaction mixture was 7.000 ppm. A control dough without the addition of the bacterial mixture was also subjected to simulated digestion. The suspension was incubated at 37 °C with stirring to artificially induce peristalsis. After 180 min of gastric digestion, the suspension was spiked with simulated intestinal fluid at pH 8.0 containing 0.1% (w / v) pancreatin and 0.15% (w / v) oxgol bile salts (Sigma-Aldrich Co.). In addition to pancreatin and bile salts, the fluid contained enzyme preparations E1, E2 (0.2 g / kg each), VeronHPP (10 g protein / 100 kg), and VeronPS (25 g protein / 100 kg) enzymes. Aspergillus oryzae (500,000 hemoglobin units / g on a tyrosine basis; enzyme 1 [E1]) and Aspergillus niger (3,000 spectrophotometric acid protease units / g; enzyme 2 [E2]), proteases routinely used in bakery applications, were supplied by BIO-CAT Inc. (Troy, VA). VeronHPP and VeronPS are bacterial proteases derived from Bacillus subtilis (AB enzymes). The enzyme mixture (E1, E2, VeronPS, VeronHPP) was not added to the control dough. Enteric digestion was carried out at 37 °C under stirring conditions (approximately 200 rpm) for 48 h. After digestion, samples were placed on ice and the concentration of hydrolyzed gluten was measured using the R5 antibody-based sandwich and competitive ELISA (R5-ELISA)

[22] according to the AOAC (Association of Official Agricultural Chemists) Method of Chemical Analysis (OMC) (Method number: AACCI38-55.01).R5-ELISA analysis was performed using the RIDASCREEN® Gliadin Competitive Detection Kit according to the manufacturer's instructions (R-Biopharm AG, Germany). In addition, an ELISA Systems Gluten Residue Detection Kit (Windsor, Australia) was used for quantification of residual gluten. The presence of epitopes in digested samples was monitored after 6, 16, 24, 36 and 48 hours of incubation through HPLC analysis. Liquid chromatography coupled with nano-electrospray ionization-ion trap tandem mass spectrometry (nano-ESI-MS / MS) was also used to confirm gluten hydrolysis and the absence of toxic epitopes.

[0064] As estimated by R5-ELISA (AOAC Chemical Analysis Method, Method Number: AACCI38-55.01), hydrolyzed gluten concentrations after 6 h of digestion ranged from 810 ± 0.02 ppm in the control to 310 ± 0.06 ppm in mix 3 (Table 2). After 16 and 24 h of digestion, the gluten content was above 100 ppm in most mixes. Importantly, after 36 h of digestion in mix 4 and mix 16, the gluten fragment levels were below 20 ppm. On the other hand, in the case of mix 4, mix 5, mix 6, mix 8 and mix 16, no gluten fragments were present at the end of incubation (48 h).

[0065] Concerning residual gluten, most of the mixes (MC1-9, 16) were below the critical threshold of 20 ppm within 24 h of digestion. Moreover, mixes 4-9 and 16 were able to reduce residual gluten to below 20 ppm within 16 h. Most importantly, mix 4 showed complete degradation after 16 h of digestion (Figure 5). MC8 and MC16 completely degraded gluten already within the first 6 h of digestion. Overall, MC4, MC8, and MC16 most efficiently removed intact gluten as well as fragmented gluten (Table 2).

[0066] [Table 2-1]

[0067] [Table 2-2]

[0068] Table 2: Concentrations (ppm) of residual gluten and peptide fragments of prolamins after 6, 16, 24, 36 and 48 h of simulated gastrointestinal digestion estimated by specific ELISA tests. Control: dough digested without bacterial cells and commercial enzymes. MC1-MC16: microbial consortia constructed using live and lysed cells of selected Lactobacillus (L.) and Bacillus (B.) strains and with E1, E2, VeronPS and VeronHPP commercial enzymes. Data are the average of three independent analyses. In the same row, superscripts differ a-j The values ​​are significantly different (P<0.05).

[0069] With respect to the calculated initial amount of gluten in the reaction mixture of 7.000 ppm, all mixtures were able to reduce the residual gluten by at least 94% after 6 hours, at least 98% after 16 hours, and at least 99.1% after 48 hours (compared to a reduction of about 84% in the control). With respect to gluten fragments, all mixtures reduced it by at least 91% after 6 hours, at least 95% after 16 hours, and at least 97% after 48 hours (compared to a reduction of about 88% in the control).

[0070] Concerning residual gluten, the most efficient strains MC4, MC8 and MC16 were able to reduce it by at least 97% after 6 h, at least 99.8% after 16 h and up to 100% after 24 h. Concerning gluten fragments, the most efficient strains MC4, MC8 and MC16 reduced it by at least 94% after 6 h, at least 97% after 16 h, at least 98% after 36 h and up to 100% after 48 h. Figure 4 shows the RP-HPLC peptide profiles of wheat bread samples digested with control (panel A), mix 4 (panel B) and mix 7 (panel C). M4 and M7 were combined with E1, E2, VeronPS and VeronHPP commercial enzymes. Mix 4 resulted in complete (93%) hydrolysis of all immunogenic peptides, whereas mix 7 only partially hydrolyzed (56%). In conclusion, we found fully functional mixes containing only 4 to 7 selected strains compared to the more extensive mixes disclosed in Example 3.

[0071] An exemplary microbial consortium was tested with and without the addition of commercial enzymes. The consortium alone significantly reduced residual and hydrolyzed gluten, and this was further enhanced with the addition of enzymes.

[0072] Experimental Example 5: Immunogenicity evaluation of gluten digests using duodenal explants from celiac disease patients The immunogenicity of digesta was estimated ex vivo by testing cytokine expression in duodenal biopsy specimens from patients with celiac disease (CD). All CD patients expressed the HLA-DQ2 phenotype. CD was diagnosed according to the European Society of Paediatric Gastroenterology, Hepatology and Nutrition guidelines for the diagnosis of celiac disease

[23] . Immediately after resection, all biopsy specimens were placed in ice-cold medium (RPMI 1640; Gibco-Invitrogen, UK) and transported to the laboratory within 30 min. Duodenal biopsy specimens were cultured for 4 h using the organ tissue culture method originally described by Browning and Trier

[24] . Briefly, biopsies were placed villous side up on a stainless steel mesh and placed on the central well of an organ tissue culture dish (Falcon, USA). The wells contained RPMI supplemented with 15% fetal bovine serum (Gibco-Invitrogen) and 1% penicillin-streptomycin. The dishes were placed in anaerobic jars and incubated at 37°C.

[0073] Control dough (positive control) (wheat bread digested without the addition of bacterial cells and microbial enzymes), mixture 4 (wheat bread digested with the addition of live and lysed cells of L. plantarum (Lactiplantibacillus plantarum) DSM 33363 and DSM 33364, L. paracasei (Lacticaseibacillus paracasei) DSM 33373, Bacillus subtilis DSM 33298, and Bacillus pumilus DSM 33301, and with E1, E2, VeronPS, and VeronHPP commercial enzymes), and mixture 7 (wheat bread digested with the addition of live and lysed cells of L. plantarum (Lactiplantibacillus plantarum) DSM 33363 and DSM 33364, L. paracasei (Lacticaseibacillus paracasei) DSM 33373, Bacillus subtilis DSM 33298, and Bacillus pumilus DSM 33301, and with E1, E2, VeronPS, and VeronHPP commercial enzymes). Live and lysed cells of Lactobacillus reuteri DSM 33362 and DSM 33366, Lactobacillus reuteri DSM 33374, L. plantarum DSM 33370, Bacillus megaterium DSM 33356, and Bacillus subtilis DSM 33353, as well as digested samples of wheat bread (digested with E1, E2, VeronPS, and VeronHPP commercial enzymes) were subjected to gliadin and glutenin polypeptide extraction and used to evaluate their ability to induce cytokine expression in duodenal biopsy specimens from CD patients.Four biopsy specimens from each CD patient were cultured in media under five conditions: (i) Mixture 4 (viable and lysed cells of L. plantarum ( Lactiplantibacillus plantarum ) DSM 33363 and DSM 33364, L. paracasei ( Lacticasei Bacillus paracasei ) DSM 33373, Bacillus subtilis DSM 33298, and Bacillus pumilus DSM 33301, together with E1, E2, Ve, and E3); (ii) mixture 7 (L. plantarum (Lactiplastobacillus plantarum) DSM 33370, DSM 33362 and DSM 33366, Lactobacillus reuteri (Lactiplastobacillus reuteri) DSM 33374, Bacillus megaterium DSM 33356, and DSM 33366); (iii) digest dough containing Mixture 16 (wheat bread digested with live and lysed cells of L. plantarum (Lactiplastobacillus plantarum) DSM 33363 and DSM 33364, Lactobacillus reuteri (Lactobacillus reuteri) DSM 33374, Bacillus megaterium DSM 33330, and Bacillus pumilus DSM 33297 and DSM 33355, with the addition of live and lysed cells of E1, E2, VeronPS, VeronHPP commercial enzymes) for 48 hours; (iv) digest control dough for 48 hours (control); and (v) medium (RPMI 1640 + gastrointestinal fluid, negative control). Biopsy specimens from each patient were rinsed and stored in RNAlater (Qiagen GmbH, Germany) at -80°C to preserve RNA. Total RNA was extracted from tissues using the RNeasy mini kit (Qiagen GmbH) according to the manufacturer's instructions. The concentration of mRNA was estimated by measuring UV absorbance at 260 nm. An aliquot of total RNA (500 ng) was reverse transcribed using random hexamers, TaqMan reverse transcription reagents (Applied Biosystems, Monza, Italy), and 3.125 U / μL of MultiScribe reverse transcriptase to a final volume of 50 μL. cDNA samples were stored at -20°C.RT-PCR was performed in 96-well plates using an ABI Prism 7500HT rapid sequence detection system (Applied Biosystems). Data collection and analysis were performed using the machine software. PCR primers and fluorogenic probes for the target genes (IFN-γ, IL-2, and IL-10) and endogenous control (the gene encoding glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) were purchased as TaqMan gene expression assays and pre-developed TaqMan assays (Applied Biosystems), respectively. The assays (analytes) were procured as a 20x mixture of PCR primers and TaqMan minor groove binder 6-carboxyfluorescein dye-labeled probes with a non-fluorescent quencher at the 3' end. Two-step reverse transcription PCR was performed using first-strand cDNA with a final concentration of 1x TaqMan gene expression assay mix and 1x TaqMan universal PCR master mix. The final reaction volume was 25 μL. Each sample was analyzed in triplicate, and all experiments were repeated twice. A no-template control (RNase-free water) was included in every plate. The following thermal cycler conditions were used: 50 °C for 2 min (uracil DNA glycosylase activation); 95 °C for 10 min; 40 cycles of 95 °C for 15 s and 60 °C for 1 min. First, standard curve and validation experiments were performed for each primer / probe set. Six serial dilutions (20–0.1 ng / μL) of IFN-γ, IL-2, or IL-10 cDNA were used as templates for each primer / probe set. Standard curves were generated by plotting threshold cycle (CT) values ​​against the logarithm of the amount of input cDNA. The CT value is the PCR cycle at which an increase in the fluorescent reporter above baseline levels is first detected. The mean values ​​of the target genes were normalized using an endogenous reference gene (GAPDH gene). Healthy duodenal biopsy specimens were used to calibrate all experiments.Levels of IFN-γ, IL-2, and IL-10 proteins secreted into the supernatants were quantified by ELISA in 96-well round-bottom plates (Tema Ricerca, Milan, Italy) according to the manufacturer's recommendations.

[0074] As expected, duodenal biopsy specimens cultured in the positive control had significantly (P<0.05) higher expression of interleukin 2 (IL-2), interleukin 10 (IL-10) (B), and interferon gamma (IFN-γ) mRNA than the negative control (RPMI1640 + gastric and intestinal fluids) (Figure 5). Compared to the negative control, samples digested with Mix 4 and Mix 16 showed similar levels (P>0.05) of IL-2, IL-10, and IFN-γ. Mix 7 synthesized less IL-2 than the positive control, but higher IL-2 synthesis compared to the negative control as well as Mix 4 and Mix 16. Similar trends were observed for IL-10 and IFN-γ. These results correlate well with the complete and partial clearance of immunogenic peptides by Mix 4 and Mix 7, respectively, as shown in Figure 4A-C.

[0075] Figure 5A shows the concentration (ng / μL) of interleukin 2 (IL-2) in duodenal biopsy specimens from CD patients. Control: wheat bread digested without the addition of bacterial cells and microbial enzymes; RPMI + gastric and intestinal fluids: negative control; microbial consortium 4: wheat bread digested with live and lysed cells of L. plantarum (Lactiplantibacillus plantarum) DSM33363 and DSM33364, L. paracasei (Lacticaseibacillus paracasei) DSM33373, Bacillus subtilis DSM33298 and Bacillus pumilus DSM33301, and with the addition of E1, E2, VeronPS, VeronHPP commercial enzymes; microbial consortium 7: L. plantarum (Lactiplantibacillus plantarum) DSM33362, DSM33366 and DSM33370, L. reuteri (Limoci) Wheat bread digested with live and lysed cells of Lactobacillus reuteri DSM 33374, Bacillus megaterium DSM 33356, Bacillus subtilis DSM 33353, and E1, E2, VeronPS, and VeronHPP commercial enzymes; and wheat bread digested with live and lysed cells of microbial consortium 16: L. plantarum (Lactiplantibacillus plantarum) DSM 33363, DSM 33364, L. paracasei (LacticaseiBacillus paracasei) DSM 33373, L. reuteri (LemociLactobacillus reuteri) DSM 33374, Bacillus megaterium DSM 33330, Bacillus pumilus DSM 33297, DSM 33355. CD1-CD10 are duodenal biopsy specimens from patients with celiac disease.

[0076] Figure 5B shows the concentration (ng / μL) of interleukin 10 (IL-10) in duodenal biopsy specimens from CD patients. The samples and microbial consortia are the same as in Figure 5A.

[0077] Figure 5C shows the concentration (ng / μL) of interferon gamma (IFN-γ) in duodenal biopsy specimens from CD patients. The samples and microbial consortia are the same as in Figure 5A.

[0078] The findings of the present invention provide evidence that selected combinations of probiotic bacterial strains can improve gluten digestion in gluten-sensitive patients and hydrolyze immunogenic peptides during gastrointestinal digestion, thereby reducing gluten toxicity to gluten-sensitive patients in general and CD patients in particular.

[0079] The following strain mixtures were identified by the present screening process according to the present invention: - L. plantarum (Lactiplantibacillus plantarum) DSM 33370, DSM 33363, DSM 33364, DSM 33365; L. paracasei (Lacticaseibacillus paracasei) DSM 33373; L. brevis (Levilactobacillus brevis) DSM 33377; Bacillus pumilus DSM 33297, DSM 33355, Bacillus licheniformis DSM 33354, Bacillus megaterium DSM 33300 and Bacillus subtilis DSM 33353, or - L. plantarum (Lactiplantibacillus plantarum) DSM 33362, DSM 33367, DSM 33368; L. paracasei (Lacticaseibacillus paracasei) DSM 33375; L. sanfrancisensis (Fructylactobacillus sanfrancisensis) DSM 33379; Bacillus pumilus DSM 33301, Bacillus megaterium DSM 33300, DSM 33356, Bacillus subtilis DSM 33298 and DSM 33353, or - L. plantarum (Lactiplantibacillus plantarum) DSM 33366 and DSM 33369, Lactobacillus reuteri (Limocylactobacillus reuteri) DSM 33374; L. paracasei (Lacticaseibacillus paracasei) DSM 33376; Pediococcus pentosaceus DSM 33371; L. sanfrancisensis (Fructilactobacillus sanfrancisensis) DSM 33378; Bacillus licheniformis DSM 33354, Bacillus pumilus DSM 33301, Bacillus megaterium DSM 33300, DSM 33356 and Bacillus subtilis DSM 33298, - L. plantarum ( Lactiplantibacillus plantarum ) DSM 33370, DSM 33363 and DSM 33364, L. paracasei ( Lacticaseibacillus paracasei ) DSM 33373, L. brevis ( Leviractobacillus brevis ) DSM 33377, Bacillus pumilus DSM 33297, DSM 33355, DSM 33301; - L. plantarum ( Lactiplantibacillus plantarum ) DSM 33362 and DSM 33367, DSM 33368, L. paracasei ( Lacticaseibacillus paracasei ) DSM 33375, Bacillus subtilis DSM 33298, Bacillus licheniformis DSM 33354, and Bacillus megaterium DSM 33300; - L. plantarum (Lactiplantibacillus plantarum) DSM 33366, DSM 33369, Lactobacillus reuteri (Limocylactobacillus reuteri) DSM 33374, L. paracasei (Lacticaseibacillus paracasei) DSM 33376, Pediococcus pentosaceus DSM 33371, Bacillus megaterium DSM 33356, and Bacillus subtilis DSM 33353; - L. plantarum ( Lactiplantibacillus plantarum ) DSM 33363 and DSM 33364, L. paracasei ( Lacticaseibacillus paracasei ) DSM 33373, Bacillus subtilis DSM 33298 and Bacillus pumilus DSM 33301; - L. brevis (Reviractobacillus brevis) DSM 33377, Pediococcus pentosaceus DSM 33371, L. plantarum (Lactiplantibacillus plantarum) DSM 33369, Bacillus pumilus DSM 33297 and Bacillus megaterium DSM 33300; - L. paracasei (LacticaseiBacillus paracasei) DSM 33375, L. plantarum (Lactiplantibacillus plantarum) DSM 33367, DSM 33368; Bacillus pumilus DSM 33355, and Bacillus licheniformis DSM 33354; - L. plantarum (Lactiplantibacillus plantarum) DSM 33370, DSM 33362 and DSM 33366, Lactobacillus reuteri (Limocylactobacillus reuteri) DSM 33374, Bacillus megaterium DSM 33356 and Bacillus subtilis DSM 33353; - L. plantarum (Lactiplantibacillus plantarum) DSM 33363, DSM 33364, L. paracasei (Lacticaseibacillus paracasei) DSM 33375, L. reuteri (Limocilactobacillus reuteri) DSM 33374, B. megaterium DSM 33300, B. pumilus DSM 33297; - L. paracasei (Lacticaseibacillus paracasei) DSM 33375, L. plantarum (Lactiplantibacillus plantarum) DSM 33367, L. reuteri (Limocylactobacillus reuteri) DSM 33374, B. megaterium DSM 33300, B. pumilus DSM 33297, B. licheniformis DSM 33354; - L. plantarum ( Lactiplantibacillus plantarum ) DSM 33363, DSM 33364, DSM 33370, L. brevis ( Leviractobacillus brevis ) DSM 33377, B. pumilus DSM 33297, Bacillus megaterium DSM 33356; - L. plantarum ( Lactiplantibacillus plantarum ) DSM 33362, DSM 33367, DSM 33368, L. paracasei ( Lacticaseibacillus paracasei ) DSM 33375, B. megaterium DSM 33300, Bacillus subtilis DSM 33353; - L. plantarum ( Lactiplantibacillus plantarum ) DSM 33366, DSM 33369, L. reuteri ( Lactiplantibacillus reuteri ) DSM 33374, L. paracasei ( Lacticaseibacillus paracasei ) DSM 33376, P. pentosaceus DSM 33371, B. pumilus DSM 33297, DSM 33355; - L. brevis (Reviractobacillus brevis) DSM 33377, P. pentosaceus DSM 33371, L. sanfrancisensis (Fructylactobacillus sanfrancisensis) DSM 33379, B. megaterium DSM 33300, B. pumilus DSM 33297; - L. plantarum (Lactiplantibacillus plantarum) DSM 33368, L. paracasei (Lacticaseibacillus paracasei) DSM 33375, L. sanfrancisensis (Fructylactobacillus sanfrancisensis) DSM 33378, B. megaterium DSM 33300, B. pumilus DSM 33297, B. licheniformis DSM 33354; - L. plantarum (Lactiplantibacillus plantarum) DSM 33362, DSM 33366, DSM 33370, L. reuteri (Rimosyllactobacillus reuteri) DSM 33374, L. sanfrancisensis (Fructylactobacillus sanfrancisensis) DSM 33378, DSM 33379, B. licheniformis DSM 33354, B. subtilis DSM 33353; - L. plantarum (Lactiplantibacillus plantarum) DSM 33363, DSM 33364, L. paracasei (Lacticaseibacillus paracasei) DSM 33373, L. reuteri (Limocilactobacillus reuteri) DSM 33374, B. megaterium DSM 33300, B. pumilus DSM 33297, DSM 33355.

[0080] A preferred combination is: - L. plantarum (Lactiplantibacillus plantarum) DSM 33363 and DSM 33364, L. paracasei (Lacticaseibacillus paracasei) DSM 33373, Bacillus subtilis DSM 33298, and Bacillus pumilus DSM 33301, or - L. plantarum (Lactiplantibacillus plantarum) DSM 33363 and DSM 33364, L. paracasei (Lacticaseibacillus paracasei) DSM 33375, Lactobacillus reuteri (Limocylactobacillus reuteri) DSM 33374, Bacillus megaterium DSM 33300, and Bacillus pumilus DSM 33297, or - L. plantarum (Lactiplantibacillus plantarum) DSM 33363 and DSM 33364, L. paracasei (Lacticaseibacillus paracasei) DSM 33373, Lactobacillus reuteri (Limocylactobacillus reuteri) DSM 33374, Bacillus megaterium DSM 33300, Bacillus pumilus DSM 33297, Bacillus pumilus DSM 33355 It is.

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Claims

1. A method for identifying a consortium of probiotic strains for enhancing the degradation of gluten and gluten-derived peptides (epitopes), The following steps: 1) providing a library of at least 10 probiotic bacterial strains; 2) culturing the probiotic bacterial strains of step 1) for at least 30 minutes under simulated gastric conditions (pH 1-4) and for at least 30 minutes under simulated intestinal conditions (pH 5.5-8.5), and selecting strains that show a CFU reduction of less than 2 log after the simulated gastrointestinal conditions; 3) measuring the proteinase activity against gluten of the strains selected in step 2) and selecting a strain capable of reducing the initial gluten level of at least 5000 ppm by 10 to 70%; 4) measuring the activities of peptidases of the strain selected in step 3), namely, aminopeptidase N type (PepN); PepI, PepO, prolyl endopeptidyl peptidase (PepP); and PepX, and selecting a strain having a peptidase activity of at least 1 U / g for at least one of these peptidases; 5) combining at least two strains selected in step 4) into a consortium of probiotic strains having an activity of the peptidases PepN, PepI, PepO and PepX of at least 1 U / g for each peptidase; 6) measuring the peptidase activity of the consortium of step 5) against the 12-mer peptide QLQPFPQPQLPY (Seq-ID No: 1), the 14-mer peptide PQPQLPYPQPQSFP (Seq-ID No: 2), the 20-mer peptide QQLPQPQQPQQSFPQQQRPF (Seq-ID No: 3), and the 33-mer peptide LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (Seq-ID No: 4), and selecting a consortium having peptidase activity that degrades all four epitopes by more than 50%; 7) measuring the peptidase activity of the consortium selected in step 6) for hydrolyzing gluten at an initial concentration of at least 5000 ppm under simulated gastric conditions (pH 1-4) for at least 30 minutes and under simulated intestinal conditions (pH 5.5-8.5) for at least 30 minutes, and selecting consortiums that reduce the initial gluten level of at least 5000 ppm and reduce the concentration of hydrolyzed residual gluten to less than 200 ppm; The method includes:

2. The following steps: 8) measuring the hydrolysis of gluten during digestion of wheat bread (1-100 g of wheat bread) by the mixture of strains selected in step 6) under simulated gastrointestinal conditions, and selecting strains that can degrade the gluten content of the wheat bread to less than 20 ppm within 6-24 hours and that do not contain gluten-derived epitopes (12-mer peptide, 14-mer peptide, 20-mer peptide, and 33-mer peptide) after 180 minutes of simulated intestinal digestion; 9) measuring the immunogenicity of the mixture of strains selected in step 7) by measuring the expression of cytokines interleukin 2 (IL-2), interleukin 10 (IL-10) and interferon gamma (IFN-γ) after 6-48 hours of culture under gastrointestinal conditions using small intestinal tissue explants from CD patients and selecting strains whose immunogenicity is equal to or lower than the negative control; The method of claim 1 , further comprising one or more of:

3. The gastric conditions in step 1) and step 7) include culturing the strain in simulated gastric fluid containing pepsin (0.5-6 g / L) at a temperature of 35° C.-39° C. for 30 minutes-300 minutes at a pH of 1-4; The intestinal conditions in step 1) include culturing the strain in simulated intestinal fluid containing pancreatin (0.02-0.6% w / v) and bile salts (0.05-0.6%) at a temperature of 35° C.-39° C. for 30-300 minutes at a pH of 5.5-8.5; The method according to claim 1 or claim 2.

4. In the step 4), the activity of peptidases, aminopeptidase N type (PepN); PepI, PepO, prolyl endopeptidyl peptidase (PepP); PepX, and PepQ peptide hydrolases, is measured using a strain in the form of a living cell or a cytoplasmic extract thereof, the strain density being 7.0-11.0 logCUF / mL and having a peptide substrate having an amino acid sequence suitable for detecting the activity of aminopeptidase N type (PepN); PepI, PepO, prolyl endopeptidyl peptidase (PepP); PepX, and PepQ peptide hydrolases. The method according to any one of claims 1 to 3.

5. The peptidase activity of step 5 is measured using live cells or cytoplasmic extracts thereof in a buffered medium (pH 6.0-9.0) at 35° C.-39° C. for 1-12 hours; A strain capable of degrading all four epitopes by more than 95% will be selected. The method according to any one of claims 1 to 4.

6. The simulated gastrointestinal conditions of step 8) include: Cultivation of the selected strains from step 6) at a strain density of 7.0-11.0 logCUF / mL; their cytoplasm; and / or Bacillus protease in simulated gastric fluid containing pepsin (0.5-6 g / L) at a temperature of 36.5° C.-37° C. for 30-300 minutes at a pH of 2-4; Cultivation of the strain selected in step 6), its cytoplasm, and / or Bacillus protease in simulated intestinal fluid containing pancreatin (0.02-0.6% w / v) and bile salts (0.05-0.6%) at a temperature of 36.5° C.-37° C. for 30 minutes to 48 hours at a pH of 7.0-8.5; The method of claim 2, comprising:

7. 7. The method according to any one of claims 1 to 6, wherein the bacterial strain is derived from one or more of the following: soil; cereals; processed cereals; sourdough; human, pig, dog, cat, rat or mouse faeces; human, pig, dog, cat, rat or mouse gastrointestinal tract specimens.

8. The method according to any one of claims 1 to 7, wherein the bacterial strain is selected from one or more of the genera Lactobacillus, Bacillus, Pediococcus and Weissella.

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