Probiotic strains for the treatment of irritable bowel syndrome and FODMAP intolerance

Abstract

The present invention relates to a probiotic strain, Lactobacillus plantarum (Lactiplantibacillus plantarum) DSM 33363, alone or in combination with other strains, such as Lactobacillus plantarum DSM 33364, Lactobacillus paracasei (Lacticaseibacillus paracasei) DSM 33373, Lactobacillus reuteri (Limosilactobacillus reuteri) DSM 33374, Bacillus megaterium (Priestia megaterium) DSM 33300, Bacillus pumilus (Bacillus megaterium) DSM 33300, Bacillus pumilus (Bacillus pumilus) DSM 33301, Bacillus pumilus (Bacillus pumilus) DSM 33302, Bacillus pumilus (Bacillus pumilus) DSM 33303, Bacillus pumilus (Bacillus pumilus) DSM 33304, Bacillus pumilus (Bacillus pumilus) DSM 33305, Bacillus pumilus (Bacillus pumilus) DSM 33306, Bacillus pumilus (Bacillus pumilus) DSM 33307, Bacillus pumilus (Bacillus pumilus) DSM 33308, Bacillus pumilus (Bacillus pumilus) DSM 33309, Bacillus pumilus (Bacillus pumilus) DSM 33310, Bacillus pumilus (Bacillus pumilus) DSM 33311, Bacillus pumilus (Bacillus pumilus) DSM 33312, Bacill The present invention relates to a preparation containing Bacillus pumilus DSM 33297 strain and Bacillus pumilus DSM 33355 in the form of live cells or a cytoplasmic extract thereof.

Description

[Technical Field]

[0001] The present invention relates to probiotic strains, including Lactobacillus plantarum (Lactiplantibacillus plantarum) DSM 33363 and Lactobacillus plantarum DSM 33364, Lactobacillus paracasei (Lacticaseibacillus paracasei) DSM 33373, Lactobacillus reuteri (Limosilactobacillus reuteri) DSM 33374, Bacillus megaterium (Priestia megaterium) DSM 33300, and Bacillus pumilus (Bacillus pumilus) DSM 33301, for use in the prevention and treatment of irritable bowel syndrome (IBS) and fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAP) intolerance in humans and animals. The present invention relates to a preparation comprising at least one bacterium selected from Bacillus pumilus DSM 33297 and Bacillus pumilus DSM 33355, or a cytoplasmic extract thereof. [Background technology]

[0002] IBS, also known as gut-brain interaction disorder, is a highly prevalent gastrointestinal motility disorder affecting up to 9% of the general population in the United States, Canada, and the United Kingdom (NPL 1) and characterized by recurrent abdominal pain and bowel problems. IBS significantly impairs the quality of life of affected individuals and places a significant burden on healthcare systems worldwide, highlighting the need for novel, effective means to treat and prevent this disease. According to the American College of Gastroenterology's clinical guidelines for the management of IBS, consensus treatment strategies include a low-FODMAP diet, treatment of constipation and diarrhea symptoms with chloride channel activators / guanylate cyclase activators, and rifaximin, respectively, and the use of gut-directed psychotherapy to treat overall IBS symptoms (NPL 2). Notably, these clinical guidelines demonstrate that treatments are symptomatic and that there is currently no cure for IBS. IBS is particularly challenging to treat due to its multifactorial nature, including disturbances in the gut microbiota, intestinal motility due to imbalances in neurotransmitter levels and function, visceral hypersensitivity, gut-brain interactions, and impaired gut barrier integrity.

[0003] Nutritional treatment strategies primarily involve exclusionary diets such as FODMAP-free, gluten-free, and dairy-free diets (Non-Patent Document 3), which only partially address the pathogenesis of this disease due to the difficulty of adherence and questionable efficacy (Non-Patent Document 4). Furthermore, probiotics from the genera Lactobacillus, Bifidobacterium, and Bacillus coagulans have shown varying efficacy when administered as assessed in a randomized controlled trial (Non-Patent Document 5). The primary rationale for using probiotics in this context is to target IBS-associated microbiota disturbances (Non-Patent Documents 6 and 7), and several publications have been published on the subject.

[0004] Recently, the taxonomic classification of some species of the genera Lactobacillus and Bacillus has been updated (Non-Patent Documents 8-10). Among them, the following species are relevant in the context of the present invention:

[0005] [Table 1] For convenience, the older names may be used in the examples, and both names may be used in the general description and claims.

[0006] Patent Document 1 discloses a preparation containing fiber and polyethylene glycol in combination with various common probiotic strains of the genera Lactobacillus, Bifidobacterium, and Bacillus as a treatment for IBS, but does not disclose whether or how the mentioned probiotics themselves affect the disease. Patent Document 2 relates to a composition containing bacteria of the genera Lactobacillus and Bifidobacterium, including Lactobacillus plantarum, Lactobacillus paracasei, and Lactobacillus reuteri, for treating IBS. Non-Patent Document 5 evaluates a low-FODMAP diet and probiotics as possible treatments for alleviating IBS symptoms, mentioning, for example, Bacillus coagulans and Lactobacillus individually, but not the possible combination of the two. Non-Patent Document 7 proposes another evaluation of existing clinical trials conducted with IBS patients, suggesting the combination of Lactobacillus rhamnosus and Lactobacillus acidophilus as particularly promising. Compositions of Bacillus megaterium and polyunsaturated fatty acid salts, and their use in, for example, the treatment of IBS, are disclosed in Non-Patent Document 11 and Patent Document 3. In Non-Patent Document 12, the Lactobacillus reuteri ATCC 55730 strain is evaluated in a study using IBS patients. Similarly, in Non-Patent Document 13, a combination of Lactobacillus paracasei HA-196 strain and Bifidobacterium R0175 strain is evaluated in IBS patients. To the inventors' knowledge, the use of Bacillus megaterium and Bacillus pumilus strains, alone or in combination with any other (Bacillus or non-Bacillus) probiotic strains, for the management and / or prevention of IBS has not been disclosed.

[0007] Many other patent applications disclose synbiotic compositions, typically defined as a combination of probiotics and prebiotics, as a possible treatment for IBS and / or other chronic gastrointestinal disorders. For example, U.S. Patent No. 5,629,993 discloses a composition of fermented cereals with Lactobacillus plantarum 299 strain for the treatment of IBS. U.S. Patent No. 5,629,993 discloses a synbiotic composition comprising a cereal-derived prebiotic and a Lactobacillus strain for use in the treatment of IBS.

[0008] A limitation of the above strategies is that the causal relationship between intestinal dysbiosis and IBS is still unclear; intestinal dysbiosis, or rather the composition of the intestinal microbiota, is influenced by many other dietary and endogenous (e.g., genetic) factors, and the impact of probiotics in this context depends on their viability and the composition of the resident microbiota. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent No. 12001603 [Patent Document 2] International Publication No. 2017 / 212433 [Patent Document 3] International Publication No. 2020 / 109474 [Patent Document 4] European Patent No. 10182284 [Patent Document 5] International Publication No. 2011 / 078781 [Non-patent literature]

[0010] [Non-Patent Document 1] Palsson, OS, Whitehead, W., Tornblom, H., Sperber, AD & Simren, M. Prevalence of Rome IV Functional Bowel Disorders Among Adults in the United States, Canada, and the United Kingdom. Gastroenterology 158, 1262-1273 e1263 (2020) [Non-patent document 2] Lacy, BE, et al. ACG Clinical Guideline: Management of Irritable Bowel Syndrome. Am J Gastroenterol 116, 17-44 (2021). [Non-patent document 3] Lenhart, A., et al. Effect of Exclusion Diets on Symptom Severity and the Gut Microbiota in Patients With Irritable Bowel Syndrome. Clin Gastroenterol Hepatol 20, e465-e483 (2022). [Non-patent document 4] Dionne, J., et al. A Systematic Review and Meta-Analysis Evaluating the Efficacy of a Gluten-Free Diet and a Low FODMAPs Diet in Treating Symptoms of Irritable Bowel Syndrome. Am J Gastroenterol 113, 1290-1300 (2018). [Non-Patent Document 5] Xie , CR , et al. Low FODMAP Diet and Probiotics in Irritable Bowel Syndrome: A Systematic Review With Network Meta-analysis. Front Pharmacol 13, 853011 (2022).

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[0011] It was an object of the present invention to provide a probiotic composition that survives gastric and small intestinal conditions for the prevention and treatment of IBS and IBS-related symptoms. [Means for solving the problem]

[0012] The present invention is based on a more rational selection, development, and application of probiotic strains that target multiple features of IBS, including gut microbiota dysbiosis. The inventors believed that an effective treatment strategy should target multiple pathophysiological features of IBS, be readily available, and be compatible with the patient's lifestyle. In WO 2021 / 129998 and in Non-Patent Document 14, the inventors disclosed the combination of Lactobacillus plantarum DSM 33363, Lactobacillus plantarum DSM 33364, Lactobacillus paracasei DSM 33373, Lactobacillus reuteri DSM 33374, Bacillus megaterium DSM 33300, Bacillus pumilus DSM 33297 and Bacillus pumilus DSM 33355 (= IBS Consortium; also called Consortium), among other combinations (Non-Patent Document 3), providing novel compounds and their ability to completely digest gluten and their use for treating so-called gluten-dependent IBS. However, it should be noted that although the clinical picture may be similar, the underlying mechanisms of so-called gluten-dependent IBS are significantly different from "true" IBS; that is, the symptoms of gluten-dependent IBS are caused by peptides derived from partial gluten digestion that have toxic and / or immunogenic activity. In fact, the majority of IBS patients do not respond to a gluten-free diet, while gluten dependence indicates either celiac disease, non-celiac gluten / wheat sensitivity, or wheat allergy (Non-Patent Document 15). Therefore, gluten dependence is an important distinguishing factor in the diagnosis, pathogenesis, and treatment of IBS.

[0013] Unexpectedly, the inventors discovered that the IBS consortium possesses functionalities that make it an effective treatment for IBS, a subtype unrelated to gluten intake and / or gastrointestinal metabolism. These functionalities include the production of the neurotransmitter gamma-aminobutyric acid (GABA) from various food matrices (whole bread, white bread, and wheat flour) during simulated gastrointestinal digestion. GABA is a key regulator of intestinal motility and has visceral antinociceptive functions (Non-Patent Documents 16 and 17). Its levels are reduced in IBS patients (Non-Patent Document 18). GABA production by the IBS consortium was several-fold stronger than in control conditions and other probiotic consortia.

[0014] Furthermore, the inventors have found that strains of the IBS consortium are able to grow on various types of FODMAPs, particularly the fructans inulin and fructooligosaccharides (FOS), thus degrading them before they reach the large intestine and can cause the typical symptoms of IBS. Finally, the IBS consortium beneficially modulates gut barrier function and survival as determined in vitro. In a gastrointestinal model, we found that digestion of wheat-based foods (white bread, wholemeal bread, wheat flour) impaired gut barrier integrity, but this was not restored by the addition of digestive proteases or (alternative) microbial consortia, whereas the IBS consortium completely restored the adverse effects of (partially) digested foods.

[0015] Thus, the present inventors have now unexpectedly discovered that their previously disclosed consortium of probiotic strains also possesses unique and surprising functions, including the release of GABA, the digestion of FODMAPs, and the protection of gastrointestinal barrier integrity. The present inventors' probiotic strains exhibit very good survival in simulated gastric and small intestinal conditions and very good storage stability, making this consortium a promising new ingredient for preparations (nutraceuticals, functional foods, and drugs) for treating and / or preventing IBS and FODMAP intolerance. [Brief explanation of the drawings]

[0016] [Figure 1] Figure 1 shows the production of gamma-aminobutyric acid (GABA) by single probiotic strains upon digestion of different foods. Panel A shows the production of GABA from white wheat bread by different strains. Panel B shows the production of GABA by Lp. plantarum DSM 33363 prepared by digestion of gluten (G), whole wheat bread (WB), MRS medium (MRS), and MRS supplemented with glutamic acid (MRS+glut). [Figure 2]FIG. 1 shows the production of gamma-aminobutyric acid (GABA) by different probiotic consortia and proteases upon digestion of different foods. [Figure 3] FIG. 1 shows that probiotic strains increase fecal concentrations of GABA in humans. [Figure 4] FIG. 1 shows growth of probiotic strains in FODMAP-supplemented minimal medium. [Figure 5] Figure 1 shows the delta (Δ) concentrations of short-chain fatty acids (acetic acid, propanoic acid) found after incubation of each strain in low-glucose control medium supplemented with inulin or FOS and the same medium without FODMAPs: MC12 (microbial consortium 12, including strains marked with grey dots); MC16 (microbial consortium 16 = IBS consortium). [Figure 6] FIG. 1 shows the evolution of TEER values ​​(ohms / cm2) as a ratio of starting values ​​by different probiotic consortia following the digestion of different foods. [Figure 7] FIG. 1 shows the evolution of TEER values ​​(ohms / cm 2 ) as a ratio of the starting values ​​from the IBS consortium before digestion. [Figure 8] Digested dough (containing 10 grams of gluten (CG)) or 100 grams of white wheat and whole wheat bread (CB and CWB, respectively) with microbial consortia MC12 and MC16 or enzyme controls. Panels show heat maps with clustering of samples (control and experiments digested with MC12 and MC16) and variables (FAA and ammonia concentrations) based on high (black) or low (white) score values. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention therefore relates to a preparation comprising the probiotic strain Lactobacillus plantarum DSM33363 for use in a method for the treatment or prevention of irritable bowel syndrome (IBS). Therefore, the present invention also provides a method for treating or preventing irritable bowel syndrome (IBS), which comprises administering a preparation containing Lactobacillus plantarum strain DSM33363. Preferably, the IBS is unrelated to gluten ingestion and / or gastrointestinal metabolism and / or physiological responses to gluten or fragmented gluten. In one embodiment of the invention, the preparation comprises one or more probiotic strains of Lactobacillus plantarum strain DSM 33364, Lactobacillus paracasei (Lacticaseibacillus paracasei) strain DSM 33373, Lactobacillus reuteri (Limosilactobacillus reuteri) strain DSM 33374, Bacillus megaterium (Priestia megaterium) strain DSM 33300, Bacillus pumilus strain DSM 33297 and Bacillus pumilus strain DSM 33355. In a preferred embodiment, the preparation comprises an IBS consortium comprising Lactobacillus plantarum strain DSM 33363, Lactobacillus plantarum strain DSM 33364, Lactobacillus paracasei strain DSM 33373, Lactobacillus reuteri strain DSM 33374, Bacillus megaterium strain DSM 33300, Bacillus pumilus strain SM33297 and Bacillus pumilus strain DSM 33355.

[0018] The therapeutic benefits of these preparations further include amelioration of IBS symptoms such as recurrent abdominal pain, bowel problems, constipation, diarrhea, flatulence and bloating. The therapeutic benefits also include amelioration of intolerance to FODMAPs. The therapeutic effect of the preparation may be due to one or more of the following modes of action: a) The preparation induces the formation of GABA from various food components in the gastrointestinal tract. The source of the food components may be, for example, wheat as well as other grain-based foods. b) The preparation digests FODMAPs such as inulin and FOS in the upper digestive tract, thereby preventing them from reaching the large intestine and causing IBS symptoms. c) The preparation improves the barrier function of the intestinal surface. This is achieved by:

[0019] In one embodiment, the preparation according to the invention is for use in the treatment or prevention of symptoms associated with IBS, preferably abdominal pain and bowel disorders. In one embodiment, the preparation according to the invention is for use in the treatment or prevention of intolerance to fermentable oligo-, di-, mono- and polyols (FODMAP intolerance). The cells of the strains according to the invention may be present in the compositions of the invention as spores (dormant), as vegetative cells (growing), as transitional cells (transitioning from vegetative to spore or vice versa), as cell extracts, or as a combination of at least two of these types of cells. In preferred embodiments, the probiotic strains are present as dormant or vegetative cells. Alternatively, in some embodiments, cytoplasmic extracts, cell-free supernatants, or heat-killed biomass of the probiotic strains are used. Alternatively, in some embodiments, the preparation further comprises one or more probiotic strains.

[0020] In a more preferred embodiment, the preparation further comprises one or more purified microbial proteases from the following: Aspergillus niger, Aspergillus oryzae, Bacillus sp., Lactobacillus sp., Pediococcus sp., Weissella sp., Rothia mucilaginosa, Rothia aeria, subtilisins, or nattokinase. Alternatively, in some embodiments, the preparation further comprises one or more enzymes that facilitate the digestion of carbohydrates, proteins, peptides, or lipids. In a preferred embodiment, the preparation for use further comprises a substance, preferably alginate, which acts as a permeabilizer for the cell membrane of microorganisms included in the genera Bacillus, Lactobacillus, Pediococcus and Weissella. Alternatively, in some embodiments, one or more probiotic strains selected from Bacillus and Lactobacillus species are immobilized, either singly or as a consortium. Immobilization can be achieved as entrapment in a porous matrix such as polysaccharide gels such as alginate, k-carrageenan, agar, chitosan, and polygalacturonic acid, or other polymer matrices such as gelatin, collagen, and polyvinyl alcohol, or on a solid surface such as cellulose and chitosan, by aggregation and microencapsulation or electrospray techniques.

[0021] In another preferred embodiment, the preparation is for treating or preventing intolerance to fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs), including fructose intolerance, in a subject in need thereof. In another embodiment, one or more probiotic strains selected from the genus Bacillus or Lactobacillus are immobilized, either alone or as a consortium.

[0022] One subject of the present invention is the use of the preparation according to the invention as a food, feed supplement, functional food, food preparation or pharmaceutical. Preferred food products according to the present invention are cereals, gummies, bread, muesli, muesli bars, health bars, biscuits, chocolate, yogurt or spreads and dairy products. Thus, in a preferred embodiment, the preparation is formulated as a pill, capsule, tablet, granular powder, operable, soluble granules, bag, pill or drinkable vial for oral administration, or formulated as a syrup or drink, or added to food, cereal, gummies, bread, muesli, muesli bars, health bars, biscuits, chocolate, yogurt or spreads. A further subject of the present invention is also the use of the preparations according to the invention as synbiotic ingredients in foods. A further subject of the present invention is a food composition comprising a preparation according to the invention and at least one further ingredient, preferably selected from proteins, carbohydrates, fats, further probiotics, prebiotics, enzymes, vitamins, immunomodulators, milk replacers, minerals, amino acids, anticoccidial agents, acid-based products, pharmaceuticals and combinations thereof. Food compositions according to the present invention also include dietary supplements in the form of, for example, pills, capsules, tablets, powders, sachets, operables, soluble granules, bags or drinkable vials, syrups, drinks, or other liquids. In another preferred embodiment, the preparation is used during the manufacture of food products derived from cereals wheat, oats, rye, barley to reduce the FODMAP content of said food products. [Example]

[0023] Probiotic strains that are resistant to gastrointestinal conditions The probiotic strains were incubated at an initial cell density of 10 9The seven strains listed in Table 1, exposed to simulated gastric and small intestinal conditions as described in Non-Patent Document 14, in terms of CFU / ml, showed very good survival under both test conditions (Non-Patent Document 14), standing out against the majority of the total of 504 strains tested, thereby verifying that the probiotic strains that are part of the present invention are reliably and sustainably effective in humans under gastric and small intestinal conditions. Table 1: Survival of probiotic strains in simulated gastric and small intestinal conditions

[0024] [Table 2] [Example]

[0025] GABA production from food by probiotic strains under simulated gastrointestinal conditions Quantitative assessment of food digestion (wheat flour, white bread, whole wheat bread) and GABA under simulated gastrointestinal conditions Three foods (wheat flour, white bread, and whole wheat bread) with and without added probiotic strains were subjected to simulated gastrointestinal conditions as described in Non-Patent Document 14. The gluten, white bread, and whole wheat bread digestion samples were assayed for GABA content in the pH 4.6 soluble nitrogen fraction using a Biochrom 30 Series Amino Acid Analyzer (Biochrom Ltd., Cambridge Science Park, England) equipped with a sodium cation exchange column (20 × 0.46 cm internal diameter). A range of GABA concentrations (Sigma Chemical Co., Milan, Italy) was used as a standard. 5% (vol / vol) cold solid sulfosalicylic acid was added, the samples were kept at 4°C for 1 hour, and then centrifuged at 15,000 × g for 15 minutes to precipitate proteins and peptides in the samples. The supernatant was filtered through a 0.22 μm pore size filter and diluted with sodium citrate (0.2 M, pH 2.2) loading buffer as needed. Amino acids were post-column derivatized with ninhydrin reagent and detected by absorbance at 440 (proline and hydroxyproline) or 570 (all other amino acids and GABA) nm. The ability of the strains to synthesize GABA was further assayed by MRS with and without glutamate. This function was also tested after gastrointestinal digestion of gluten, white bread, and whole wheat bread.

[0026] result All strains tested, along with Lactobacillus plantarum, produced significantly higher concentrations of GABA, with strain DSM33363 producing significantly higher levels, approximately sixfold higher on average, than all other strains. Therefore, we next tested this strain under additional conditions to determine how different substrates—wheat flour, whole wheat bread, and gluten prepared from MRS medium—were used for GABA production. As shown in panel B, Lactobacillus plantarum strain DSM33363 was able to produce GABA from all substrates. Figure 1 shows the production of gamma-aminobutyric acid (GABA) by a single probiotic strain upon digestion of different foods. Panel A shows the production of GABA from white bread by different strains. Panel B shows the production of GABA by L. plantarum DSM33363 upon digestion of gluten (G), whole wheat bread (WB), MRS medium (MRS), and MRS supplemented with glutamic acid (MRS+glut). Similarly, the GABA-producing capacity of an IBS consortium containing L. plantarum DSM 33363 was compared with other consortia (L. plantarum DSM 33366, L. reuteri DSM 33374; L. paracasei DSM 33376; Pediococcus (P.) pentosaceus DSM 33371; Bacillus pumilus DSM 33297 and DSM 33355) and with two different protease preparations. This difference was independent of the substrate and was a unique characteristic of the IBS consortium. Figure 2 shows the gamma-aminobutyric acid (GABA) production by different probiotic consortia and proteases upon digestion of different foods. [Example]

[0027] Fecal levels of GABA when supplemented with probiotic strains versus placebo Overview of human studies assessing the impact of gluten challenge Healthy human subjects aged 18-50 years were administered either probiotic (N = 50) or placebo (N = 20) capsules, one capsule per day for a total of 34 days, followed by a 7-day washout period. Probiotic capsules contained a total of at least 3 × 10 9 The IBS consortium was included in the study at levels of ≥ CFU, as confirmed by periodic testing throughout the study. Fecal samples were collected at various time points for GABA quantification.

[0028] result FIG. 3 shows that probiotic strains increase fecal concentrations of GABA in humans. GABA concentrations were measured in fecal samples from eight volunteers (four placebo-treated and four probiotic-treated). These samples were collected at baseline (day 0), after 34 days of daily intake of one capsule filled with probiotic strains (day 34), and after a seven-day washout period. Fecal GABA concentrations in the placebo group significantly decreased over the course of the study. In contrast, in the verum arm, GABA concentrations increased approximately sevenfold after 34 days of probiotic treatment. Seven days after ablation, GABA concentrations returned to near baseline levels. [Example]

[0029] Ability of probiotic strains to use FODMAPs as growth substrates Growth of probiotic strains in FODMAP-supplemented minimal growth medium Low-glucose medium (LBG with 0.1 g / L glucose for Bacillus species and MRS with 2 g / L glucose for Lactobacillus species) was used as the minimal growth medium and was supplemented with or without 5 g / L FODMAP (β-glucan, FOS, or inulin) compared to a control medium with a high glucose content of 20 g / L. Strains were cultured for 24 h, and CFU concentrations were determined by plate counting with serial dilutions. Quantification of SCFAs One mL of the culture supernatant (obtained after centrifugation at 10,000 rpm for 10 minutes) spiked with 10 μL of 33 ppm internal standard solution (4-methyl-2-pentanol) was placed in a 20 mL glass vial and sealed with a polytetrafluoroethylene (PTFE)-coated silicone rubber septum (20 mm diameter) (Supelco, Bellefonte, PA, USA). A microextraction procedure was performed, and the extracted compounds were desorbed at 220 °C for 3 minutes in splitless mode. The Clarus 680 (Perkin Elmer) gas chromatograph was equipped with an Rtx-WAX capillary column (30 m x 0.25 mm id, 0.25 μm film thickness) (Restek, Bellfonte, PA, USA). The column temperature was initially 35 °C for 8 minutes, then ramped to 60 °C at 4 °C / min, to 160 °C at 6 °C / min, and finally to 200 °C at 20 °C / min, where it was held for 15 minutes. Helium was used as the carrier gas at a flow rate of 1 mL / min. A single quadrupole mass spectrometer, Clarus SQ8C (Perkin Elmer), was connected to the gas chromatography system. The source and transfer line temperatures were maintained at 250 and 230 °C, respectively. Electron ionization mass was recorded at 70 eV with an m / z (mass-to-charge ratio) interval of 34–350. Chromatograms generated by GC-MS showed peaks representing individual compounds. Each chromatogram was analyzed for peak identification using the National Institute of Standards and Technology 2008 (NIST) library. A peak area threshold of >1,000,000 and a match probability of 90% or greater were used for VOC identification, followed by manual visual inspection of the fragmentation patterns. Quantitative data for identified compounds were obtained by interpolation of their relative areas to the internal standard area.

[0030] result FIG. 4 shows the growth of probiotic strains in FODMAP-supplemented minimal medium. All test strains grew in low-glucose medium (CM-LG) supplemented with inulin (inu) or FOS to levels similar to those in the high-glucose control medium (CM), whereas β-glucan supplementation (CM-LG-β) generally had no growth-stimulating effect. As shown in Figure 1, strains DSM33363 and DSM33374 were highly efficient at metabolizing inulin and FOS, respectively, as evidenced by enhanced acetate formation. Figure 5 shows the delta (Δ) concentrations of short-chain fatty acids (acetic acid, propanoic acid) found after incubation of each strain in control medium containing low glucose supplemented with inulin or FOS, and the same medium without FODMAPs: MC12 (microbial consortium 12, including strains marked with gray dots); MC16 (microbial consortium 16 = IBS consortium). [Example]

[0031] Effect of food digested under simulated gastrointestinal conditions on intestinal barrier integrity and its modulation by probiotic strains Preparation of Caco-2 cells for membrane integrity assays on transwell plates The Caco-2 cell line was obtained from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Braunschweig, Germany) ACC169. Cells were cultured in MEM (Sigma Aldrich #51416C) supplemented with 2 mM L-glutamine, 10% (v / v) FBS, 1% (v / v) non-essential amino acids, and 1% (v / v) penicillin / streptomycin. Caco-2 cells were grown in 25 cm 2 and / or 75cm 2 The cells were grown in T-flasks and maintained at 37°C in a humidified atmosphere of 5% CO2 and 2-95% air. They were subcultured every 3-4 days at 80-90% confluence. When near confluence, the cells were detached with trypsin, counted, and plated at 1 x 10 per 500 μl onto 12 mm polycarbonate membrane Transwell inserts (Corning®) with 0.4 μm pore size. 5Cells were seeded at a density of 1000 x 1000 cells / well. Cells were cultured for 21 days to achieve differentiation, with the growth medium changed every 2-3 days. TEER values ​​were monitored by a Millicell® ERS meter (Merck KGaA, Darmstadt, Germany). Values ​​were approximately 600-800 ohms / cm. 2 Monolayers were considered confluent when a plateau was reached in the reading. Confluent monolayers were then used for further experiments. Digesta samples from foods (gluten / wheat flour and white bread) containing two different probiotic consortia (IBS consortium and MC12) were investigated for their effect on barrier integrity in the Caco-2 cell monolayer assay as described above. All approaches were prepared in triplicate.

[0032] result Figure 6 shows the TEER values ​​(ohms / cm) as a ratio of the starting values ​​by different probiotic consortia upon digestion of different foods. 2 ) occurs. The residual gluten-containing, probiotic consortium-free bread control showed a complete breakdown of barrier integrity and a drop in TEER values ​​over the first 3 hours. On the other hand, the different probiotic consortia in the different foods (gluten, white bread) without residual gluten showed stable barrier integrity compared to the untreated medium control. The IBS consortium performed even better than the medium control (untreated cells) and the probiotic consortium MC12. Figure 7 shows the TEER values ​​(ohms / cm) as a ratio of the starting values ​​from the IBS consortium before digestion. 2 ) trends. The pre-digestive IBS consortium stabilizes the barrier integrity of the monolayer and therefore exerts a beneficial effect in itself. [Example]

[0033] Distinct amino acid profiles of dough digested by the IBS consortium versus MC12 or enzyme-digested dough Preparation of enzyme- or microbial consortium-digested dough and subsequent amino acid profiling Pooled saliva samples from three subjects were mixed with 10 g of gluten-enriched powder extract from wheat flour and placed in a beaker containing 10 mL of NaK buffer solution (0.05 M, pH 6.9). The mixture was then mechanically homogenized in a laboratory stomacher for 30 seconds. A microbial consortium was added to each suspension. Additionally, a control sample containing the above-mentioned commercial enzyme was prepared. Bacterial cell-free Promod™ or Tollerase® G was also included. The saliva-containing dough was added to simulated gastric fluid containing 125 mM NaCl, 7 mM KCl, 45 mM NaHCO3, and 3 g / L pepsin, while the pH was adjusted to 2 with 0.1 M HCl. The samples were incubated at 37°C under stirring conditions (200 × g) to simulate peristalsis. After 180 minutes, simulated intestinal fluid (pH 8.0) containing 0.1% (w / v) pancreatin (Sigma-Aldrich Co.) and 0.15% (w / v) bile salts (Sigma-Aldrich Co.) was added to each sample and maintained at 37°C under stirring conditions (200 × g). These conditions simulating the intestinal phase were extended for up to 48 hours (i.e., a 3-hour gastric phase and a 45-hour intestinal phase). Amino acid quantification was performed as described in Example 2, with post-column derivatization of amino acids with ninhydrin reagent and detection by absorbance at 440 nm (proline and hydroxyproline) or 570 nm (all other amino acids).

[0034] result Figure 8 shows that application of the IBS consortium (MC16) resulted in high levels of Asp, GABA, Lys, Gly, and Orn, while significantly depleting the amino acids L-histidine and L-glutamic acid, biomarkers and exacerbating factors of IBS (Non-Patent Documents 19 and 20). These surprising effects were not seen in the related consortium (MC12) or in control conditions without added bacterial cells. Digested dough (containing 10 grams of gluten (CG)) or 100 grams of white wheat and whole wheat bread (CB and CWB, respectively) with microbial consortia MC12 and MC16 or enzyme controls. Panels show heat maps with clustering of samples (control and experiments digested with MC12 and MC16) and variables (FAA and ammonia concentrations) based on high (black) or low (white) score values.

Claims

1. A preparation comprising the probiotic strain Lactobacillus plantarum (Lactiplantibacillus plantarum) DSM 33363 for use in a method for the treatment or prevention of irritable bowel syndrome (IBS).

2. 2. The preparation of claim 1, further comprising one or more probiotic strains of Lactobacillus plantarum strain DSM 33364, Lactobacillus paracasei (Lacticaseibacillus paracasei) strain DSM 33373, Lactobacillus reuteri (Limosilactobacillus reuteri) strain DSM 33374, Bacillus megaterium (Priestia megaterium) strain DSM 33300, Bacillus pumilus strain DSM 33297 and Bacillus pumilus strain DSM 33355.

3. 3. The preparation of claim 1 or 2, wherein the IBS is independent of gluten intake and / or gastrointestinal metabolism.

4. A preparation according to any one of claims 1 to 3, wherein the probiotic strains are present as dormant or vegetative cells.

5. A preparation according to any one of claims 1 to 4, wherein a cytoplasmic extract, a cell-free supernatant or a heat-killed biomass of a probiotic strain is used.

6. 6. The preparation of any one of claims 1 to 5, further comprising one or more digestive enzymes such as microbial proteases purified from Aspergillus niger, Aspergillus oryzae, Bacillus sp., Lactobacillus sp., Pediococcus sp., Weissella sp., Rothia mucilaginosa, Rothia aeria, subtilisins, nattokinase, lactase, glutenase, gliadin hydrolase, protein hydrolase, carbohydrate hydrolase, lipid hydrolase, lipase, etc.

7. The preparation according to any one of claims 1 to 6, further comprising a substance that acts as a permeabilizer for the cell membrane of microorganisms belonging to the genera Bacillus, Lactobacillus, Pediococcus, and Weissella, or an alginate.

8. A preparation according to any one of claims 1 to 7, wherein the one or more probiotic strains are immobilised singly or as a consortium.

9. The preparation according to any one of claims 1 to 8, which is a food, a feed supplement, a functional food, a processed food product or a pharmaceutical product.

10. For oral administration, it may be formulated as a pill, capsule, tablet, granular powder, operable, soluble granule, bag, pill or drinkable vial, or prepared as a syrup or drink, or Added to food, cereals, gummies, bread, muesli, muesli bars, health bars, biscuits, chocolate, yogurt or spreads, A preparation according to any one of claims 1 to 9.

11. 11. A food composition comprising the preparation of any one of claims 1 to 10 and at least one further ingredient selected from proteins, carbohydrates, fats, further probiotics, prebiotics, enzymes, vitamins, immunomodulators, milk replacers, minerals, amino acids, anticoccidial agents, acid-based products, pharmaceuticals and combinations thereof.

12. 1. A preparation comprising the probiotic strain Lactobacillus plantarum DSM 33363 for use in a method for the treatment or prevention of abdominal pain and / or bowel disorders, optionally in combination with one or more of the following probiotic strains: Lactobacillus plantarum DSM 33364, Lactobacillus paracasei DSM 33373, Lactobacillus reuteri DSM 33374, Bacillus megaterium DSM 33300, Bacillus pumilus DSM 33297 and Bacillus pumilus DSM 33355.

13. 1. A preparation comprising the probiotic strain Lactobacillus plantarum DSM 33363 for use in a method for the treatment or prevention of intolerance to fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAP intolerance), optionally in combination with one or more of the following probiotic strains: Lactobacillus plantarum DSM 33364, Lactobacillus paracasei DSM 33373, Lactobacillus reuteri DSM 33374, Bacillus megaterium DSM 33300, Bacillus pumilus DSM 33297, and Bacillus pumilus DSM 33355.

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