BACTERIAL CONSORTIUM COMPRISING AT LEAST ONE BACILLUS AND LACTOBACILLUS STRAIN FOR GLUTEN DEGRADATION
20230128187 · 2023-04-27
Assignee
Inventors
- Bodo SPECKMANN (Kahl, DE)
- Michael Schwarm (Alzenau, DE)
- Stefan PELZER (Guetersloh, DE)
- Thomas BERNGRUBER (Hamm, DE)
- Marco GOBBETTI (Bozen-Bolzano, IT)
- Raffaella DI CAGNO (Bozen-Bolzano, IT)
Cpc classification
A61K35/742
HUMAN NECESSITIES
A61P1/14
HUMAN NECESSITIES
A61K35/744
HUMAN NECESSITIES
C12R2001/125
CHEMISTRY; METALLURGY
A23L7/104
HUMAN NECESSITIES
A23L33/135
HUMAN NECESSITIES
C12R2001/01
CHEMISTRY; METALLURGY
International classification
A23L33/135
HUMAN NECESSITIES
A61K35/742
HUMAN NECESSITIES
A61K35/744
HUMAN NECESSITIES
A61P1/14
HUMAN NECESSITIES
Abstract
A preparation comprising probiotic strains belonging to the genera Bacillus sp., Lactobacillus sp., optionally Pediococcus sp. as viable cells or cytoplasmic extract thereof, and proteases, wherein the strains can degrade peptide sequences of gliadins. The preparation can be used to produce gluten-free foods from gluten-containing cereals or to treat gluten-related disorders.
Claims
1. A preparation comprising a consortia of at least one bacterial strain selected from the genus Bacillus and at least one bacterial strain selected from the genus Lactobacillus, for use in the degradation of gluten to a gluten content of 20 ppm or less, wherein the consortia of strains can degrade 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).
2. The preparation according to claim 1, wherein the consortia of strains can degrade 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) by at least 80%.
3. The preparation according to claim 1, wherein the consortia of strains can degrade the gluten to a digest that does not cause an immunogenic or toxic response in a small intestine or a small intestinal explant of a subject or an animal affected by a gluten-related disorder, and/or wherein all strains of the consortia survive (less than 2 log CFU loss) in simulated gastric (pH 1.0-4.0) and intestinal (pH 5.4-6.8, 0.05-0.6% bile acids, or a concentration of bile acids present in humans under in vivo conditions) conditions, and/or wherein members of the consortia have complementary PepP, PepO, PepX, PepI, and PepN activities with at least one peptidase activity equal to or more than 3 U/g (PepP), 5 U/g (PepO), 20 U/g (PepX), 17 U/g (PepI), and 20 U/g (PepN).
4. The preparation according to claim 1, wherein the Bacillus strains are selected from the group consisting of Bacillus pumilus, Bacillus subtilis, Bacillus licheniformis, and Bacillus megaterium.
5. The preparation according to claim 1, wherein the Lactobacillus strains are selected from the group consisting of Lactobacillus plantarum (Lactiplantibacillus plantarum), Lactobacillus casei (Lacticaseibacillus casei), Lactobacillus paracasei (Lacticaseibacillus paracasei), Lactobacillus brevis (Levilactobacillus brevis), Lactobacillus sanfranciscensis (Fructilactobacillus sanfranciscensis), and Lactobacillus reuteri (Limosilactobacillus reuteri).
6. The preparation according to claim 1, wherein the probiotic strains are present in a dormant form or as vegetative cells.
7. The preparation according to claim 1, wherein cytoplasmic extracts or cell-free supernatants or heat-killed biomass of the strains are used.
8. The preparation according to claim 1, wherein the preparation further comprises at least one selected from the group consisting of Pediococcus sp., and Weissella sp.
9. The preparation according to claim 1, wherein the preparation further comprises one or more microbial proteases purified from at least one selected from the group consisting of Aspergillus niger, Aspergillus oryzae, Bacillus sp., Lactobacillus sp., Pediococcus sp., Weissella sp., Rothia niucilaginosa, Rothia aeria, subtilisins, nattokinase, arabinoxylans, barley grain fibre, oat grain fibre, rye fibre, wheat bran fibre, inulins, fructooligosaccharides (FOS), galactooligosaccharides (GOS), resistant starch, beta-glucans, glucomannans, galactoglucomannans, guar gum, xylooligosaccharides, and alginate.
10. The preparation according to claim 1 for treating or preventing gluten-related disorders, wherein the disorder is at least one selected from the group consisting of celiac disease, non-celiac gluten sensitivity, wheat allergy, and gluten-sensitive irritable bowel syndrome in a subject or an animal in need thereof.
11. The preparation according to claim 1 for producing gluten-free foods from at least one gluten-containing cereal selected from the group consisting of wheat, barley, rye, and oat.
12. The preparation according to claim 1, further comprising a substance, which acts as a permeabilizer of a microbial cell membrane of at least one selected from the group consisting of Bacillus sp., Lactobacillus sp., Pediococcus sp., and Weissella sp.
13. The preparation according to claim 1, wherein the strains are selected from Bacillus sp., Lactobacillus sp., Pediococcus sp., and Weissella sp., and are immobilized individually or as consortia.
14. A preparation, comprising at least one strain of each of the following groups 1-5: Group 1: Lactobacillus paracasei (Lacticaseibacillus paracasei) DSM 33373, Lactobacillus paracasei (Lacticaseibacillus paracasei) DSM 33375, Group 2: Bacillus subtilis DSM 33298, Bacillus pumilus DSM 33297, Bacillus licheniformis DSM 33354, Bacillus megaterium DSM 33356, Pediococcus pentosaceus DSM 33371, Lactobacillus plantarum (Lactiplantibacillus plantarum) DSM 33370, Lactobacillus brevis (Levilactobacillus brevis) DSM 33377, Lactobacillus paracasei (Lacticaseibacillus paracasei) DSM 33376, Lactobacillus paracasei (Lacticaseibacillus paracasei) DSM 33375, Lactobacillus reuteri (Limosilactobacillus reuteri) DSM 33374, Lactobacillus plantarum (Lactiplantibacillus plantarum) DSM 33367, Lactobacillus plantarum (Lactiplantibacillus plantarum) DSM 33363, Lactobacillus paracasei (Lacticaseibacillus paracasei) DSM 33373, Lactobacillus plantarum (Lactiplantibacillus plantarum) DSM 33362, Group 3: Lactobacillus sanfranciscensis (Fructilactobacillus sanfranciscensis) DSM 33378, Lactobacillus sanfranciscensis (Fructilactobacillus sanfranciscensis) DSM 33379, Pediococcus pentosaceus DSM 33371, Lactobacillus plantarum (Lactiplantibacillus plantarum) DSM 33370, Lactobacillus plantarum (Lactiplantibacillus plantarum) DSM 33369, Lactobacillus reuteri (Limosilactobacillus reuteri) DSM 33374, Lactobacillus plantarum (Lactiplantibacillus plantarum) DSM 33363, Lactobacillus paracasei (Lacticaseibacillus paracasei) DSM 33373, Group 4: Bacillus subtilis DSM 33353, Bacillus pumilus DSM 33355, Bacillus pumilus DSM 33301, Group 5: Bacillus megaterium DSM 33300, Lactobacillus sanfranciscensis (Fructilactobacillus sanfranciscensis) DSM 33378, Pediococcus pentosaceus DSM 33371, Lactobacillus brevis (Levilactobacillus brevis) DSM 33377, Lactobacillus plantarum (Lactiplantibacillus plantarum) DSM 33368, Lactobacillus reuteri (Limosilactobacillus reuteri) DSM 33374, Lactobacillus plantarum (Lactiplantibacillus plantarum) DSM 33367, Lactobacillus plantarum (Lactiplantibacillus plantarum) DSM 33366, Lactobacillus plantarum (Lactiplantibacillus plantarum) DSM 33364, and Lactobacillus paracasei (Lacticaseibacillus paracasei) DSM 33373.
15. The preparation according to claim 14, comprising at least three different strains.
16. The preparation according to claim 14, further comprising the strain selected at least one from the group consisting of following strains: L. plantarum (Lactiplantibacillus plantarum) DSM 33370, DSM 33363, DSM 33364; 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. sanfranciscensis (Fructilactobacillus sanfranciscensis) 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 (Limosilactobacillus reuteri) DSM 33374; L. paracasei (Lacticaseibacillus paracasei) DSM 33376; Pediococcus pentosaceus DSM 33371; L. sanfranciscensis (Fructilactobacillus sanfranciscensis) DSM 33378; Bacillus licheniformis DSM 33354, Bacillus pumilus DSM 33301, Bacillus megaterium DSM 33300, DSM 33356, and Bacillus subtilis DSM 33298, or 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 (Limosilactobacillus 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 (Limosilactobacillus reuteri) DSM 33374, Bacillus megaterium DSM 33300, Bacillus pumilus DSM 33297, Bacillus pumilus DSM 33355, or L. plantarum (Lactiplantibacillus plantarum) DSM 33370, DSM 33363, and DSM 33364, L. paracasei (Lacticaseibacillus paracasei) DSM 33373, L. brevis (Levilactobacillus brevis) DSM 33377, Bacillus pumilus DSM 33297, DSM 33355, DSM 33301, or 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, or L. plantarum (Lactiplantibacillus plantarum) DSM 33366, DSM 33369, Lactobacillus reuteri (Limosilactobacillus reuteri) DSM 33374, L. paracasei (Lacticaseibacillus paracasei) DSM 33376, Pediococcus pentosaceus DSM 33371, Bacillus megaterium DSM 33356, and Bacillus subtilis DSM 33353, or L. brevis (Levilactobacillus brevis) DSM 33377, Pediococcus pentosaceus DSM 33371, L. plantarum (Lactiplantibacillus plantarum) DSM 33369, Bacillus pumilus DSM 33297, and Bacillus megaterium DSM 33300, or L. paracasei (Lacticaseibacillus paracasei) DSM 33375, L. plantarum (Lactiplantibacillus plantarum) DSM 33367, DSM 33368; Bacillus pumilus DSM 33355, and Bacillus licheniformis DSM 33354, or L. plantarum (Lactiplantibacillus plantarum) DSM 33370, DSM 33362, and DSM 33366, Lactobacillus reuteri (Limosilactobacillus reuteri) DSM 33374, Bacillus megaterium DSM 33356, and Bacillus subtilis DSM 33353, or L. paracasei (Lacticaseibacillus paracasei) DSM 33375, L. plantarum (Lactiplantibacillus plantarum) DSM 33367, L. reuteri (Limosilactobacillus reuteri) DSM 33374, B. megaterium DSM 33300, B. pumilus DSM 33297, B. licheniformis DSM 33354, or L. plantarum (Lactiplantibacillus plantarum) DSM 33363, DSM 33364, DSM 33370, L. brevis (Levilactobacillus brevis) DSM 33377, B. pumilus DSM 33297, Bacillus megaterium DSM 33356, or L plantarum (Lactiplantibacillus plantarum) DSM 33362, DSM 33367, DSM 33368, L. paracasei (Lacticaseibacillus paracasei) DSM 33375, B. megaterium DSM 33300, B. subtilis DSM 33353, or L. plantarum (Lactiplantibacillus plantarum) DSM 33366, DSM 33369, L. reuteri (Limosilactobacillus reuteri) DSM 33374, L. paracasei (Lacticaseibacillus paracasei) DSM 33376, P. pentosaceus DSM 33371, B. pumilus DSM 33297, DSM 33355, or L. brevis (Levilactobacillus brevis) DSM 33377, P. pentosaceus DSM 33371, L. sanfranciscensis (Fructilactobacillus sanfranciscensis) DSM 33379, B. megaterium DSM 33300, B. pumilus DSM 33297, or L. plantarum (Lactiplantibacillus plantarum) DSM 33368, L. paracasei (Lacticaseibacillus paracasei) DSM 33375, L. sanfranciscensis (Fructilactobacillus sanfranciscensis) DSM 33378, B. megaterium DSM 33300, B. pumilus DSM 33297, B. licheniformis DSM 33354, or L. plantarum (Lactiplantibacillus plantarum) DSM 33362, DSM 33366, DSM 33370, L. reuteri (Limosilactobacillus reuteri) DSM 33374, L. sanfranciscensis (Fructilactobacillus sanfranciscensis) DSM 33378, DSM 33379, B. licheniformis DSM 33354, and B. subtilis DSM 33353.
17. A product comprising at least one selected from the group consisting of a food supplement, a pet food supplement, a food product, a pet food product, and a pharmaceutical product comprising the preparation of claim 14.
Description
WORKING EXAMPLES
Example 1. Probiotic Microorganisms Resistant to Gastrointestinal Conditions
[0193] Simulated gastric and intestinal fluids were used as described by Fernández et al. [19]. Stationary-phase-grown cells were harvested at 8000 g for 10 min, washed with physiologic solution, and suspended in 50 ml of simulated gastric juice (cell density of 10 log CFU/ml), which contains NaCl (125 mM/I), KCl (7 mM/I), NaHCO3 (45 mM/I), and pepsin (3 g/I) (Sigma-Aldrich CO., St. Louis, Mo., USA) [20]. The final pH was adjusted to 2.0, 3.0, and 8.0. The value of pH 8.0 was used to investigate the influence of the components of the simulated gastric juice, apart from the effect of low pH. The suspension was incubated at 37° C. under anaerobic conditions and agitation to simulate peristalsis. Aliquots of this suspension were taken at 0, 90, and 180 min, and viable count was determined. The effect of gastric digestion was also determined by suspending cells in reconstituted skimmed milk (RSM) (11% solids, w/v) before inoculation of simulated gastric juice at pH 2.0. The final pH after the addition of RSM was ca. 3.0. This condition was assayed to simulate the effect of the food matrix during gastric transit [20]. After 180 min of gastric digestion, cells were harvested and suspended in simulated intestinal fluid, which contains 0.1% (w/v) pancreatin and 0.15% (w/v) Oxgall bile salt (Sigma-Aldrich Co.) at pH 8.0. The suspension was incubated at 37° C. under agitation and aliquots were taken at 0, 90, and 180 min [21]. 119 out of <400 tested strains showed a decrease of less than 2 log of initial 1×10.sup.10 CFU/ml and defined as resistant to simulated gastrointestinal conditions.
Example 2. Protease and Peptidase Activities of Single Strains Resistant to Gastrointestinal Conditions
[0194] All 119 strains (Lactobacillus sp., 63 strains; Weissella sp., 3 strains; Pediococcus sp., 1 strain; and Bacillus sp., 51 strains) showing resistance to simulated gastrointestinal conditions were tested for their peptidase and proteinase activities towards synthetic substrates. To assay the peptidase activities, cultures of each strain from the late exponential phase of growth (ca. 9.0 log CFU/ml) were used. Aliquots (0.3 g [dry weight]) of washed cell pellets were re-suspended 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 enzymes loosely associated to the cell wall. The cytoplasmic extract was prepared by incubating bacterial suspensions with lysozyme in 50 mM Tris-HCl (pH 7.5) buffer containing 24% sucrose at 37° C. for 60 min, under stirring conditions (ca. 160 rpm). Spheroblasts were resuspended in isotonic buffer and sonicated for 40 sat 16 A/s (Sony Prep model 150; Sanyo, United Kingdom). The extracts were concentrated 10-fold by freeze-drying, re-suspended in 5 mM Tris-HCl (pH 7.0), and dialyzed for 24 h at 4° C. 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 lactobacilli were measured by 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 (Sigma Chemical Co), 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 mixture was incubated at 37° C. for 180 min, and the absorbance was measured at 410 nm. The data were compared to standard curves set up by using p-nitroaniline. One unit of activity was defined as the amount of enzyme required to liberate 1 μmol of p-nitroaniline for min under the assay conditions. Based on Principal Component Analysis (PCA) data from the above peptidase activities, some strains clearly separated from the other ones (
[0195]
[0196]
Example 3. Peptidase Activities of Mixture of Strains Against Immunogenic Epitopes
[0197] Bacillus, Lactobacillus, and Pediococcus strains showing very high peptidase activities (at least for one peptidase) were assessed as mixed strains to combine intense and complementary enzyme activities. Various mixtures were used to assay their capacity to in vitro degrade immunogenic epitopes responsible for gluten intolerance.
[0198] The hydrolysis of peptides was carried out using combinations of cytoplasmic extracts of previously selected bacteria strains. Immunogenic epitopes corresponding to fragments 57-68 (Q-L-Q-P-F-P-Q-P-Q-L-P-Y) of α9-gliadin, 62-75 (P-Q-P-Q-L-P-Y-P-Q-P-Q-S-F-P) of A-gliadin, 134-153 (Q-Q-L-P-Q-P-Q-Q-P-Q-Q-S-F-P-Q-Q-Q-R-P-F) of γ-gliadin, and 57-89 (L-Q-L-Q-P-F-P-Q-P-Q-L-P-Y-P-Q-P-Q-L-P-Y-P-Q-P-Q-L-P-Y-P-Q-P-Q-P-F) (33-mer) of α2-gliadin were chemically synthesized and used at an initial concentration of 1 mM. Hydrolysis was monitored by RP-HPLC. Single peaks from RP-HPLC were analysed by nano-ESI tandem mass spectrometry (nano-ESI-MS/MS). The mixtures of strains that showed the best hydrolysis of synthetic immunogenic epitopes were numbers 3, 4 and 5 (
[0199] Strain mixtures were as follows: [0200] 1. L. plantarum (Lactiplantibacillus plantarum) DSM 33362, DSM 33363, DSM 33364, DSM 33366; L. sanfranciscensis (Fructilactobacillus sanfranciscensis) DSM 33379; Bacillus pumilus DSM 33297, DSM 33355, Bacillus licheniformis DSM 33354, Bacillus megaterium DSM 33300, Bacillus subtilis DSM 33353. [0201] 2. L. paracasei (Lacticaseibacillus paracasei) DSM 33375, DSM 33376; L. plantarum (Lactiplantibacillus plantarum) DSM 33369, DSM 33368; L. sanfranciscensis (Fructilactobacillus sanfranciscensis) DSM 33378; Bacillus licheniformis DSM 33354, Bacillus megaterium DSM 33300, DSM 33356, Bacillus pumilus DSM 33297, DSM 33301. [0202] 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. [0203] 4. L. plantarum (Lactiplantibacillus plantarum) DSM 33362, DSM 33367, DSM 33368; L. paracasei (Lacticaseibacillus paracasei) DSM 33375; L. sanfranciscensis (Fructilactobacillus sanfranciscensis) DSM 33379; Bacillus pumilus DSM 33301, Bacillus megaterium DSM 33300, DSM 33356, and Bacillus subtilis DSM 33298, DSM 33353. [0204] 5. L. plantarum (Lactiplantibacillus plantarum) DSM 33366, DSM 33369, Lactobacillus reuteri (Limosilactobacillus reuteri) DSM 33374; L. paracasei (Lacticaseibacillus paracasei) DSM 33376; Pediococcus pentosaceus DSM 33371, L. sanfranciscensis (Fructilactobacillus sanfranciscensis) DSM 33378; Bacillus licheniformis DSM 33354, Bacillus pumilus DSM 33301, Bacillus megaterium DSM 33300, DSM 33356, Bacillus subtilis DSM 33298. [0205] 6. L. plantarum (Lactiplantibacillus plantarum) DSM 33370, DSM 33367, Lactobacillus reuteri (Limosilactobacillus reuteri) DSM 33374; L. brevis (Levilactobacillus brevis) DSM 33377; Bacillus pumilus DSM 33301, Bacillus megaterium DSM 33300, DSM 33356, Bacillus subtilis DSM 33298.
Example 4. Degradation of Gluten Under Simulated Gastrointestinal Conditions by Different Consortia
[0206] The gluten degradation under simulated gastrointestinal digestion was assessed. With the intention to develop a feasible technical solution for full degradation of gluten in vivo, we searched for minimal combinations containing as few strains as possible and as many as needed.
[0207] Using mixtures 1-6 of Example 3 as a starting point, the following consortia, selected from 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 (Levilactobacillus 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) were prepared: [0208] 1. L. plantarum (Lactiplantibacillus plantarum) DSM 33370, DSM 33363 and DSM 33364, L. paracasei (Lacticaseibacillus paracasei) DSM 33373 L. brevis (Levilactobacillus brevis) DSM 33377, Bacillus pumilus DSM 33297, DSM 33355, DSM 33301; [0209] 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; [0210] 3. L. plantarum (Lactiplantibacillus plantarum) DSM 33366, DSM 33369, Lactobacillus reuteri (Limosilactobacillus reuteri) DSM 33374, L. paracasei (Lacticaseibacillus paracasei) DSM 33376, Pediococcus pentosaceus DSM 33371, Bacillus megaterium DSM 33356, and Bacillus subtilis DSM 33353; [0211] 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; [0212] 5. L. brevis (Levilactobacillus brevis) DSM 33377, Pediococcus pentosaceus DSM 33371, L. plantarum (Lactiplantibacillus plantarum) DSM 33369, Bacillus pumilus DSM 33297 and Bacillus megaterium DSM 33300; [0213] 6. L. paracasei (Lacticaseibacillus paracasei) DSM 33375, L. plantarum (Lactiplantibacillus plantarum) DSM 33367, DSM 33368; Bacillus pumilus DSM 33355, and Bacillus licheniformis DSM 33354; [0214] 7. L. plantarum (Lactiplantibacillus plantarum) DSM 33370, DSM 33362, and DSM 33366, Lactobacillus reuteri (Limosilactobacillus reuteri) DSM 33374, Bacillus megaterium DSM 33356, and Bacillus subtilis DSM 33353. [0215] 8. L. plantarum (Lactiplantibacillus plantarum) DSM 33363, DSM 33364, L. paracasei (Lacticaseibacillus paracasei) DSM 33375, L. reuteri (Limosilactobacillus reuteri) DSM 33374, B. megaterium DSM 33300, B. pumilus DSM 33297; [0216] 9. L. paracasei (Lacticaseibacillus paracasei) DSM 33375, L. plantarum (Lactiplantibacillus plantarum) DSM 33367, L. reuteri (Limosilactobacillus reuteri) DSM 33374, B. megaterium DSM 33300, B. pumilus DSM 33297, B. licheniformis DSM 33354; [0217] 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; [0218] 11. L plantarum (Lactiplantibacillus plantarum) DSM 33362, DSM 33367, DSM 33368, L. paracasei (Lacticaseibacillus paracasei) DSM 33375, B. megaterium DSM 33300, B. subtilis DSM 33353; [0219] 12. L. plantarum (Lactiplantibacillus plantarum) DSM 33366, DSM 33369, L. reuteri (Limosilactobacillus reuteri) DSM 33374, L. paracasei (Lacticaseibacillus paracasei) DSM 33376, P. pentosaceus DSM 33371, B. pumilus DSM 33297, DSM 33355; [0220] 13. L. brevis (Levilactobacillus brevis) DSM 33377, P. pentosaceus DSM 33371, L. sanfranciscensis (Fructilactobacillus sanfranciscensis) DSM 33379, B. megaterium DSM 33300, B. pumilus DSM 33297; [0221] 14. L. plantarum (Lactiplantibacillus plantarum) DSM 33368, L. paracasei (Lacticaseibacillus paracasei) DSM 33375, L. sanfranciscensis (Fructilactobacillus sanfranciscensis) DSM 33378, B. megaterium DSM 33300, B. pumilus DSM 33297, B. licheniformis DSM 33354; [0222] 15. L. plantarum (Lactiplantibacillus plantarum) DSM 33362, DSM 33366, DSM 33370, L. reuteri (Limosilactobacillus reuteri) DSM 33374, L. sanfranciscensis (Fructilactobacillus sanfranciscensis) DSM 33378, DSM 33379, B. licheniformis DSM 33354, B. subtilis DSM 33353; [0223] 16. L. plantarum (Lactiplantibacillus plantarum) DSM 33363, DSM 33364, L. paracasei (Lacticaseibacillus paracasei) DSM 33373, L. reuteri (Limosilactobacillus reuteri) DSM 33374, B. megaterium DSM 33300, B. pumilus DSM 33297, DSM 33355.
[0224] Five grams of wheat bread (chewed for 30 s and collected in a beaker with 10 mL of NaK-phosphate 0.05 M, pH 6.9) or related dough were suspended in simulated gastric juice 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 of the pooled selected strains as live (with a final cell density of approximately 9.0 log CFU/mL) and 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 addition of bacterial mixture, was also subjected to simulated digestion. The suspension was incubated at 37° C., under stirring to simulate peristalsis. After 180 min of gastric digestion, the suspension was added with simulated intestinal fluid, which contained 0.1% (w/v) pancreatin and 0.15% (w/v) Oxgall bile salt (Sigma-Aldrich Co.) at pH 8.0. Besides pancreatin and bile salt, the fluid contained enzymatic preparation E1, E2 (each at 0.2 g/kg), Veron HPP (10 g/100 kg of protein) and Veron PS (g/100 kg of protein) enzymes. Proteases of Aspergillus oryzae (500,000 haemoglobin units on the tyrosine basis/g; enzyme 1 [E1]) and Aspergillus niger (3,000 spectrophotometric acid protease units/g; enzyme 2 [E2]), routinely used for bakery applications, were supplied by BIO-CAT Inc. (Troy, Va.). Veron HPP and Veron PS are bacterial proteases from Bacillus subtilis (AB Enzymes). Enzymatic mixture (E1, E2, Veron PS, Veron HPP) was not added in the control dough.
[0225] Intestinal digestion was carried out for 48 h at 37° C. under stirring conditions (ca. 200 rpm). After digestion, samples were put on ice and the concentration of hydrolysed gluten was determined according to a AOAC (Association of Official Agricultural Chemists) International Official Method of Analysis (OMA) (Method No. AACCI 38-55.01) using R5 antibody-based sandwich and competitive ELISA (R5-ELISA) [22]. R5-ELISA analysis was carried out with the RIDASCREEN® Gliadin competitive detection kit according to the instructions of the manufacturer (R-Biopharm AG, Germany). Moreover, 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 h 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 the hydrolysis of gluten and the absence of toxic epitopes.
[0226] As estimated by the R5-ELISA (AOAC Official Method of Analysis, Method No. AACCI 38-55.01), after 6 h of digestion the concentration of hydrolysed gluten was in the range of 810±0.02 ppm for the control and 310±0.06 ppm for mixture 3 (Table 2). After 16 and 24 h of digestion, gluten content was 100 ppm for most of the mixtures, with the exception of mixture 16. Importantly, gluten fragment levels were below 20 ppm after 36 h of digestion with mixtures 4 and 16; while gluten fragments were completely absent at the end of incubation (48 h) for mixture 4, 5, 6, 8, and 16.
[0227] Regarding the residual gluten, most of the mixtures (MC1-9, 16) reduced it below the critical threshold of 20 ppm within 24 h of digestion. Furthermore, mixtures 4-9 and 16 were able to decrease residual gluten to ≤20 ppm within 16 h. Most importantly, the mixture 4 showed complete after 16 h of digestion (
TABLE-US-00003 TABLE 2 Concentration (ppm) of residual gluten and peptide fragments of prolamins after 6, 16, 24, 36 and 48 h of simulated gastrointestinal digestion, as estimated by a specific ELISA tests. Control: dough digested without bacterial cells and commercial enzymes; MC1-MC16: Microbial consortia constructed by using live and lysed cells of selected Lactobacillus (L.) and Bacillus (B.) strains and E1, E2, Veron PS, Veron HPP commercial enzymes. Data are the mean of three independent analyses. Sandwich ELISA assay (Residual gluten) Competitive ELISA assay (Gluten fragments) Strains 6 h 16 h 24 h 36 h 48 h 6 h 16 h 24 h 36 h 48 h Control 1100.sup.a ± 620.sup.a ± 367.sup.a ± 256.sup.a ± 75.sup.a ± 810.sup.a ± 400.sup.a ± 397.sup.a ± 381.sup.a ± 375.sup.a ± 0.06 0.09 0.05 0.04 0.06 0.03 0.02 0.08 0.07 0.05 MC1 L. plantarum DSM33370, DSM33363, 406.sup.b ± 135.sup.b ± 19.sup.e ± 0.sup.e 0.sup.e 310.sup.f ± 250.sup.d ± 200.sup.e ± 170.sup.e ± 65.sup.g ± DSM33364; L. paracasei DSM33373; 0.04 0.06 0.01 0.05 0.03 0.04 0.02 0.01 L. brevis DSM33377; B. pumilus DSM33297, DSM33355, DSM33301 MC2 L. plantarum DSM33362, DSM33367, 346.sup.c ± 121.sup.b ± 15.sup.e ± 0.sup.e 0.sup.e 332.sup.f ± 226.sup.ef ± 167.sup.f ± 158.sup.e ± 150.sup.c ± DSM33368; L. paracasei DSM33375; 0.07 0.03 0.01 0.05 0.04 0.03 0.02 0.02 B. subtilis DSM33298; B. licheniformis DSM33354; B. megaterium DSM33300 MC3 L. plantarum DSM33366, DSM33369; 382.sup.c ± 99.sup.c ± 12.sup.f ± 0.sup.e 0.sup.e 315.sup.f ± 272.sup.d ± 256.sup.d ± 244.sup.c ± 228.sup.b ± L. reuteri DSM33374; L. paracasei 0.03 0.02 0.01 0.06 0.07 0.04 0.05 0.02 DSM33376; P. pentosaceus DSM33371; B. megaterium DSM33356; B. subtilis DSM33353 MC4 L. plantarum DSM33363, DSM33364; 190.sup.cd ± 0.sup.g 0.sup.g 0.sup.e 0.sup.e 399.sup.e ± 233.sup.e ± 112.sup.g ± 0.sup.j 0.sup.g L. paracasei DSM33373; B. subtilis 0.05 0.08 0.07 0.05 DSM33298; B. pumilus DSM33301 MC5 L. brevis DSM33377; P. pentosaceus 380.sup.b ± 18.sup.g ± 5.sup.g ± 0.sup.e 0.sup.e 398.sup.e ± 221.sup.e ± 154.sup.fg ± 46.sup.i ± 0.sup.g DSM33371; L. plantarum DSM33369; 0.06 0.01 0.01 0.04 0.05 0.03 0.02 B. pumilus DSM33297; B. megaterium DSM33300 MC6 L. paracasei DSM33375; L. plantarum 350.sup.c ± 15.sup.e ± 2.sup.g ± 0.sup.e 0.sup.e 404.sup.e ± 245.sup.de ± 100.sup.g ± 79.sup.h ± 0.sup.g DSM33367, DSM33368; B. pumilus 0.06 0.02 0.01 0.06 0.05 0.08 0.04 DSM33355; B. licheniformis DSM33354 MC7 L. plantarum DSM33370, DSM33362, 360.sup.c ± 20.sup.e ± 10.sup.f ± 0.sup.e 0.sup.e 401.sup.e ± 261.sup.d ± 150.sup.f ± 99.sup.g ± 78.sup.g ± DSM33366; L. reuteri DSM33374; B. 0.09 0.06 0.01 0.07 0.05 0.03 0.04 0.05 megaterium DSM33356; B. subtilis DSM33353 MC8 L. plantarum DSM33363, DSM33364; 18.sup.g ± 3.sup.g ± 0.sup.g 0.sup.e 0.sup.e 323.sup.f ± 228.sup.e ± 218.sup.e ± 157.sup.e ± 0.sup.g L. paracasei DSM33375; L. reuteri 0.03 0.01 0.08 0.06 0.05 0.06 DSM33374; B. megaterium DSM33300; B. pumilus DSM33297 MC9 L. paracasei DSM33375; L. plantarum 60.sup.f ± 12.sup.f ± 0.sup.g 0.sup.e 0.sup.e 319.sup.f ± 211.sup.f ± 196.sup.ef ± 195.sup.de ± 152.sup.c ± DSM33367; L. reuteri DSM33374; B. 0.04 0.01 0.06 0.05 0.03 0.07 0.02 megaterium DSM33300; B. pumilus DSM33297; B. licheniformis DSM33354 MC10 L. plantarum DSM33363, DSM33364, 112.sup.e ± 77.sup.d ± 70.sup.d ± 0.sup.e 0.sup.e 465.sup.d ± 370.sup.b ± 243.sup.de ± 145.sup.ef ± 97.sup.f ± DSM33370; L. brevis DSM33377; B. 0.06 0.04 0.02 0.09 0.06 0.05 0.04 0.03 pumilus DSM33297; B. megaterium DSM33356 MC11 L. plantarum DSM33368, DSM33362, 221.sup.d ± 89.sup.c ± 69.sup.d ± 50.sup.d ± 43.sup.d ± 512.sup.c ± 367.sup.b ± 340.sup.b ± 300.sup.b ± 123.sup.de ± DSM33367; L. paracasei DSM33375; 0.05 0.07 0.06 0.04 0.03 0.06 0.08 0.09 0.06 0.05 B. megaterium DSM33300; B. subtilis DSM33353 MC12 L. plantarum DSM33366, DSM33369; 145.sup.e ± 110.sup.c ± 89.sup.c ± 75.sup.c ± 63.sup.b ± 601.sup.b ± 312.sup.c ± 289.sup.c ± 288.sup.b ± 143.sup.ed ± L. reuteri DSM33374; L. paracasei 0.06 0.05 0.03 0.02 0.03 0.09 0.06 0.07 0.05 0.03 DSM33376; P. pentosaceus DSM33371; B. pumilus DSM33297, DSM33355 MC13 L. brevis DSM33377; P. pentosaceus 163.sup.de ± 122.sup.b ± 82.sup.c ± 45.sup.d ± 0.sup.e 523.sup.c ± 322.sup.c ± 321.sup.b ± 215.sup.d ± 134.sup.d ± DSM33371; L. sanfranciscensis 0.06 0.04 0.02 0.03 0.07 0.07 0.06 0.07 0.05 DSM33379; B. megaterium DSM33300; B. pumilius DSM33297 MC14 L. plantarum DSM33368; L. paracasei 234.sup.d ± 135.sup.b ± 120.sup.b ± 108.sup.b ± 56.sup.c ± 587.sup.b ± 333.sup.c ± 256.sup.d ± 211.sup.d ± 167.sup.c ± DSM33375; L. sanfranciscensis 0.08 0.07 0.07 0.05 0.03 0.09 0.09 0.08 0.08 0.07 DSM33378; B. megaterium DSM33300; B. pumilus DSM33297; B. licheniformis DSM33354 MC15 L. plantarum DSM33362, DSM33366, 199.sup.d ± 100.sup.c ± 81.sup.c ± 59.sup.d ± 40.sup.d ± 498.sup.c ± 318.sup.c ± 280.sup.c ± 256.sup.c ± 118.sup.e ± DSM33370; L. reuteri DSM33374; L. 0.05 0.04 0.05 0.04 0.03 0.08 0.04 0.03 0.08 0.05 sanfranciscensis DSM33378, DSM33379; B. licheniformis DSM33354; B. subtilis DSM33353 MC16 L. plantarum DSM33363, DSM33364; 19.sup.g ± 11.sup.f ± 0.sup.g 0.sup.e 0.sup.e 280.sup.g ± 200.sup.f ± 50.sup.h ± 10.sup.j ± 0.sup.g L. paracasei DSM33373; L. reuteri 0.03 0.01 0.06 0.05 0.03 0.01 DSM33374; B. megaterium DSM33300; B. pumilus DSM33297, DSM33355 .sup.a-jValues with different superscript letters, in the same row, differ significantly (P < 0.05).
[0228] Based on the calculated initial amount of gluten in the reaction mixture of 7.000 ppm, regarding the residual gluten, all the mixtures were able to reduce it by at least 94% after 6 h (in comparison to a reduction of around 84% for the control), by at least 98% after 16 h and up to at least 99.1% after 48 h. Regarding gluten fragments, those were reduced by all mixtures by at least 91% after 6 h (in comparison to a reduction of around 88% for the control), by at least 95% after 16 h and up to at least 97% after 48 h.
[0229] Regarding the 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. Regarding the gluten fragments, those were reduced by the most efficient strains MC4, MC8, and MC16 by at least 94% after 6 h, by at least 97% after 16 h, by at least 98% after 36 h and to 100% after 48 h.
[0230] For exemplary microbial consortia we performed experiments with and without added commercial enzymes. The consortia alone led to strong reductions of residual as well as hydrolysed gluten, and this was further enhanced by added enzymes.
Example 5. Assessment of Immunogenicity of Gluten Digests by Using Duodenal Explants from Celiac Disease Patients
[0231] Immunogenicity of the digests was ex vivo estimated by testing the 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 European Society for Paediatric Gastroenterology, Hepatology, and Nutrition criteria [23]. Immediately after excision, all biopsy specimens were placed in ice-chilled culture 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, the biopsy specimens were oriented villous side up on a stainless-steel mesh and positioned over the central well of an organ tissue culture dish (Falcon, USA). The well contained RPMI supplemented with 15% foetal calf serum (Gibco-Invitrogen) and 1% penicillin-streptomycin (Gibco-lnvitrogen, UK). Dishes were placed into an anaerobic jar and incubated at 37° C.
[0232] Digested samples of 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 E1, E2, Veron PS, Veron HPP commercial enzymes) and Mixture 7 (wheat bread digested with the addition of live and lysed cells of L. plantarum (Lactiplantibacillus plantarum) DSM 33362, and DSM 33366, Lactobacillus reuteri (Limosilactobacillus reuteri) DSM 33374, L. plantarum (Lactiplantibacillus plantarum) DSM 33370, Bacillus megaterium DSM 33356, and Bacillus subtilis DSM 33353 and E1, E2, Veron PS, Veron HPP commercial enzymes) were subjected to gliadin and glutenin polypeptide extraction and used for assessing their ability to induce cytokine expression in duodenal biopsy specimens from CD patients. Four biopsy specimens from each CD patient were cultured with culture medium under five conditions: (i) with doughs containing the 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 E1, E2, Veron PS, Veron HPP commercial enzymes) digested for 48 h; (ii) with dough containing Mixture 7 (wheat bread digested with the addition of live and lysed cells of L. plantarum (Lactiplantibacillus plantarum) DSM 33370, DSM 33362, and DSM 33366, Lactobacillus reuteri (Limosilactobacillus reuteri) DSM 33374, Bacillus megaterium DSM 33356, and Bacillus subtilis DSM 33353 and E1, E2, Veron PS, Veron HPP commercial enzymes) digested for 48 h; (iii) with dough containing Mixture 16 (wheat bread digested with the addition of live and lysed cells of L. plantarum (Lactiplantibacillus plantarum) DSM 33363 and DSM 33364, Lactobacillus reuteri (Limosilactobacillus reuteri) DSM 33374, Bacillus megaterium DSM 33330, and Bacillus pumilus DSM 33297 and DSM 33355 and E1, E2, Veron PS, Veron HPP commercial enzymes) digested for 48 h; (iv) with control dough digested for 48 h (Control); and (v) with culture medium (RPMI 1640+gastric and intestinal juice, negative control). Biopsy specimens from each patient were rinsed and stored in RNAlater (Qiagen GmbH, Germany) at −80° C. to preserve the RNA. Total RNA was extracted from the tissues using the RNeasy minikit (Qiagen GmbH) according to the manufacturer's instructions. The concentration of mRNA was estimated by determination of the UV absorbance at 260 nm. Aliquots of total RNA (500 ng) were 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. The cDNA samples were stored at −20° C. RT-PCR was performed in 96-well plates using an ABI Prism 7500HT fast sequence detection system (Applied Biosystems). Data collection and analyses were performed using the machine software. PCR primers and fluorogenic probes for the target genes (IFN-γ, IL-2, and IL-10) and the endogenous control (gene coding for glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) were purchased as a TaqMan gene expression assay and a pre-developed TaqMan assay (Applied Biosystems), respectively. The assays were supplied as a 20× mix of PCR primers and TaqMan Minor Groove Binder 6-carboxyfluorescein dye-labelled probes with a non-fluorescent quencher at the 3′ end of the probe. Two-step reverse transcription-PCR was performed using first-strand cDNA with a final concentration of 1× TaqMan gene expression assay mix and 1× TaqMan universal PCR master mix. The final reaction volume was 25 μl. Each sample was analysed in triplicate, and all experiments were repeated twice. A non-template control (RNase-free water) was included with every plate. The following thermal cycler conditions were used: 2 min at 50° C. (uracil DNA glycosylase activation), 10 min at 95° C., and 40 cycles of 15 s at 95° C. and 1 min at 60° C. Initially, a standard curve and a validation experiment were performed for each primer/probe set. Six serial dilutions (20 to 0.1 ng/μl) of IFN-γ, IL-2, or IL-10 cDNA were used as a template for each primer/probe set. A standard curve was generated by plotting the threshold cycle (CT) values against the log of the amount of input cDNA. The CT value is the PCR cycle at which an increase in reporter fluorescence above the baseline level is first detected. The average value for the target gene was normalized using an endogenous reference gene (the GAPDH gene). A healthy duodenal biopsy specimen was used to calibrate all the experiments. The levels of IFN-γ, IL-2, and IL-10 proteins secreted into the supernatant were quantified by ELISA in 96-well round-bottom plates (Tema Ricerca, Milan, Italy) according to the manufacturer's recommendations.
[0233] As expected, the duodenal biopsy specimens incubated with positive control produced 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 (RPMI 1640+gastric and intestinal juice) (
[0234]
[0235]
[0236]
[0237] The findings of this invention provide evidence that the selected combinations of probiotic bacterial strains have the potential to improve the digestion of gluten in gluten-sensitive patients and to hydrolyse immunogenic peptides during gastrointestinal digestion, which decreases gluten toxicity for gluten-sensitive patients in general, and for CD patients particularly.
REFERENCES
[0238] 1. Sapone A, Bai J C, Ciacci C, Dolinsek J, Green P H, Hadjivassiliou M, Kaukinen K, Rostami K, Sanders D S, Schumann M et al: Spectrum of gluten-related disorders: consensus on new nomenclature and classification. BMC Med 2012, 10:13. [0239] 2. Niland B, Cash B D: Health Benefits and Adverse Effects of a Gluten-Free Diet in Non-Celiac Disease Patients. Gastroenterol Hepatol (N Y) 2018, 14(2):82-91. [0240] 3. Lis D M, Stellingwerff T, Shing C M, Ahuja K D, Fell J W: Exploring the popularity, experiences, and beliefs surrounding gluten-free diets in nonceliac athletes. Int J Sport Nutr Exerc Metab 2015, 25(1):37-45. [0241] 4. 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(4):573-581 [0242] 5. Thompson T, Dennis M, Higgins L A, Lee A R, Sharrett M K: Gluten-free diet survey: are
[0243] Americans with coeliac disease consuming recommended amounts of fibre, iron, calcium and grain foods? J Hum Nutr Diet 2005, 18(3):163-169. [0244] 6. Larretxi I, Simon E, Benjumea L, Miranda J, Bustamante M A, Lasa A, Eizaguirre F J, Churruca I: Gluten-free-rendered products contribute to imbalanced diets in children and adolescents with celiac disease. Eur J Nutr 2018. [0245] 7. De Palma G, Nadal I, Collado M C, Sanz Y: Effects of a gluten-free diet on gut microbiota and immune function in healthy adult human subjects. Br J Nutr 2009, 102(8):1154-1160. [0246] 8. Galipeau H J, McCarville J L, Huebener S, Litwin O, Meisel M, Jabri B, Sanz Y, Murray J A, Jordana M, Alaedini A et al: Intestinal microbiota modulates gluten-induced immunopathology in humanized mice. Am J Pathol 2015, 185(11):2969-2982. [0247] 9. 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(4):670-683. [0248] 10. Francavilla R, Ercolini D, Piccolo M, Vannini L, Siragusa S, De Filippis F, De Pasquale I, Di Cagno R, Di Toma M, Gozzi G et al: Salivary microbiota and metabolome associated with celiac disease. Appl Environ Microbiol 2014, 80(11):3416-3425. [0249] 11. 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(2):508-518. [0250] 12. Francavilla R, Cristofori F, Verzillo L, Gentile A, Castellaneta S, Polloni C, Giorgio V, Verduci E, D'Angelo E, Dellatte S et al: Randomized Double-Blind Placebo-Controlled Crossover Trial for the Diagnosis of Non-Celiac Gluten Sensitivity in Children. Am J Gastroenterol 2018, 113(3):421-430. [0251] 13. 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(14). [0252] 14. Herran A R, Perez-Andres J, Caminero A, Nistal E, Vivas S, Ruiz de Morales J M, Casqueiro J: Gluten-degrading bacteria are present in the human small intestine of healthy volunteers and celiac patients. Res Microbiol 2017, 168(7):673-684. [0253] 15. Caminero A, Herran A R, Nistal E, Perez-Andres J, Vaquero L, Vivas S, Ruiz de Morales J M, 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(2):309-319. [0254] 16. Pyle G G, Paaso B, Anderson B E, Allen D D, Marti T, Li Q, Siegel M, Khosla C, Gray G M: Effect of pretreatment of food gluten with prolyl endopeptidase on gluten-induced malabsorption in celiac sprue. Clin Gastroenterol Hepatol 2005, 3(7):687-694. [0255] 17. Krishnareddy S, Stier K, Recanati M, Lebwohl B, Green P H: Commercially available glutenases: a potential hazard in coeliac disease. Therap Adv Gastroenterol 2017, 10(6):473-481. [0256] 18. 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(Pt 2):311-318. [0257] 19. Fernandez M F, Boris S, Barbes C: Probiotic properties of human lactobacilli strains to be used in the gastrointestinal tract. J Appl Microbiol 2003, 94(3):449-455. [0258] 20. Zarate G, Chaia A P, Gonzalez 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(9):1214-1221. [0259] 21. 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(8):792-801. [0260] 22. Valdes I, Garcia E, Llorente M, Mendez E: Innovative approach to low-level gluten determination in foods using a novel sandwich enzyme-linked immunosorbent assay protocol. Eur J Gastroenterol Hepatol 2003, 15(5):465-474. [0261] 23. of ES: Revised criteria for diagnosis of coeliac disease. Report of Working Group of European Society of Paediatric Gastroenterology and Nutrition. Arch Dis Child 1990, 65(8):909-911. [0262] 24. Browning T H, Trier J S: Organ culture of mucosal biopsies of human small intestine. J Clin Invest 1969, 48(8):1423-1432.