Probiotic compositions for modulating the gut microbiome

Abstract

The present invention relates to the use of a preparation comprising the probiotic strains 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 for treating or preventing dysbiosis in humans and animals.

Description

[Technical field]

[0001] The present invention relates to the use of a preparation comprising the probiotic strains 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 for treating or preventing dysbiosis in humans and animals.

[0002] An increasing number of health disorders (diabetes, allergic and autoimmune diseases, cancer, inflammatory bowel disease, brain disorders, etc.) have been linked to the dysfunction of the gut microbiome [1]. Since this association seems to be bidirectional, strategies targeting the microbiota have been considered as new therapeutic possibilities to prevent or treat these disorders. The gut microbiome influences human and animal physiology, for example, through soluble factors derived from microbial metabolism, modulation of local and systemic immune cells, and modulation of the enteric nervous system and the vagus nerve. On the other hand, the composition and activity of the gut microbiota are influenced by intrinsic factors (genome, sex, age, disease) and a large number of extrinsic factors, but diet is probably the most important determinant. Modulation of the composition and activity of the microbiota through diet includes the application of prebiotics, probiotics, synbiotics and antibiotics. The most studied and commercially available probiotics are microorganisms, mainly species of the genera Lactobacillus and Bifidobacterium.

[0003] Dysbiosis has been described in patients with food intolerance, for example to histamine [2] or gluten [3]. Evaluation of the gut microbiota composition in the feces and / or duodenum of patients with celiac disease (CD) compared to healthy controls revealed reduced alpha diversity, increased levels of the genera Proteobacteria, Bacteroides, Prevotella, Escherichia, Pseudomonas, Neisseria, Serratia and Haemophilus, as well as reduced levels of Streptococcus, Akkermansia, Bifidobacteria and Lactobacilli [3–6]. Recently, an increased abundance of Proteobacteria, especially Neisseria spp., has been identified in the salivary, duodenal and fecal microbiota of CD patients [ 7 – 9 ].

[0004] Dysbiosis in CD patients may develop during the course of the disease and in that sense plays a rather bystander role. However, other studies have shown that dysbiosis may precede the onset of CD and act as an exacerbating factor of the disease

[10] . This view is supported by the functional analysis of dysbiotic dominant species of the human gut microbiota (e.g., Neisseria flavescens and Pseudomonas aeruginosa) [6,8]. Similarly, the Escherichia coli ENT CAI:5 strain isolated from a fecal sample of a CD patient exacerbated gluten-induced immunopathology in clean SPF mice

[11] . Human digestive proteases only partially digest proline-rich gliadins. In general, a low capacity of this digestive enzyme limits the digestive process and generates gluten-derived peptides (epitopes), which act as triggering factors for celiac disease in susceptible individuals

[12] . Neisseria flavescens and Pseudomonas aeruginosa have the ability to increase the content of these epitopes

[13] and efficiently cross the intestinal mucosal barrier

[14] . These characteristics suggest that pathogens associated with dysbiosis play a detrimental role in the pathogenesis of celiac disease

[10] and possibly also in the pathogenesis of other gluten-related disorders such as non-celiac gluten sensitivity (NCGS). NCGS shares common features with CD in terms of symptoms and treatment (gluten-free diet) and is the second most symptomatic manifestation in the series of gluten-related disorders. Few reports exist on the composition of the gut microbiota in NCGS. Due to diagnostic imprecision in NCGS and overlapping symptoms with irritable bowel syndrome, these analyses are both fewer in number and less clear than those for CD.Nevertheless, the gut microbiota of NCGS patients generally shows decreased levels of Bifidobacteria and butyrate-producing Firmicutes [15,16], increased levels of Proteobacteria, Actinobacillus and Finegoldia, as well as a decreased abundance

[16] . A gluten-free diet (GFD) is the only available and therefore essential treatment for both NCGS and CD patients. For both diseases, the effect of a gluten-free diet on the composition of the gut microbiota has been studied. Mexican NCGS patients responded to a GFD with increased abundance of Gammaproteobacteria and Pseudomonas

[17] . Other studies have reported that NCGS treated with a GFD showed a decrease in Bacteroides, Blautia, Dorea, Coprococcus, Collinsella, and Lachnospiraceae, and an increase in Bacteroidaceae.

[18] The microbiota composition of CD patients was also (negatively) affected by the GFD, with an increase in the genera Proteobacteria, Pseudomonas, Prevotella, and Streptococcus, while the diversity and abundance of Lactobacillii and Bifidobacteria were clearly decreased.

[18] As GFD is the only treatment currently available for CD patients, its adverse side effects consequently indicate an urgent need for novel (co)therapeutic strategies modulating the microbiome.

[0005] Although the GFD has gained popularity among more than just patients suffering from gluten-related disorders and is currently one of the most sought-after elimination diets

[19] , it does have drawbacks and potential harms, especially for healthy individuals.

[20] The effects of the GFD and diets with low gluten content (up to 2 g per day) on the abundance of bacterial populations in healthy individuals have been studied and summarized

[18] . Overall, gluten removal reduced the abundance of Bifidobacteria, Lactobacillii, Faecalibacterium prausnitzii, Dorea spp. (e.g., Dorea longicatena), Blautia wexlerae, Veillonellaceae, Roseburia, Anaeostipes hadrus, and Eubacterium hallii, and reduced the abundance of E. coli. coli, Slackia, Enterobacteriaceae, Coriobacteriaceae and Proteobacteriaceae increased [18,21,22].

[0006] Overall, gut dysbiosis is associated with an increasing number of pathological conditions, including food intolerances, and although the causal relationship is often unclear, the discovery of mechanisms of action in several taxa with different prevalences makes dysbiosis a potential disease factor worth targeting. Although numerous attempts have been made to correct dysbiosis by the application of prebiotics, probiotics and synbiotics in various settings, limitations of currently available microbiome-modulating interventions are their lack of precision and unclear clinical efficacy.

[0007] In WO 2021 / 129998 and

[12] , the applicants previously disclosed various combinations of Bacillus and Lactobacillus strains and their ability to fully digest gluten. However, the prebiotic or microbiome modulating effect of these strains or combinations thereof, which is the subject of the present invention, has not been disclosed anywhere until now.

[0008] Francavilla et al. reported an increase in the abundance of putative Lactobacillus, Staphylococcus, and Bifidobacterium species in the feces of CD patients after six weeks of treatment with a composition containing Lactobacillus casei LMG 101 / 37 P-17504, Lactobacillus plantarum CECT 4528, Bifidobacterium animalis subsp. lactis Bi1 LMG P-17502, and Bifidobacterium breve Bbr8 LMG P-17501 strains

[23] . The application of other strains of the genus Bifidobacterium (B. breve, B. longum) to target the gut microbiota has been summarized, including by modulating the abundance of Firmicutes, Bacteroidetes, and Bacteroides fragilis, but the taxa encompassing these groups have not been further evaluated.

[0009] The inventors previously disclosed a multi-strain probiotic composition that completely hydrolyzes gluten to a non-toxic and non-immunogenic digesta (PCT / EP2020 / 083770,

[12] ). Surprisingly, the inventors discovered that this composition induces beneficial changes in the human gut microbiome in the context of a gluten-free or controlled gluten-containing diet. Such changes have not been previously disclosed for this or any other composition. These changes included unconventional modulation of several taxa previously associated with gluten-related disorders and gluten-free diets. The inventors' discovery of the prebiotic function of this multi-strain probiotic formulation paves the way for novel strategies to prevent, treat and / or cure disorders and conditions associated with microbiome dysfunction, especially in humans and animals following a gluten-free or gluten-reduced diet or in humans and animals suffering from celiac disease or similar gluten-related disorders.

[0010] Thus, the present invention is directed to the use of a preparation comprising 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 for treating or preventing dysbiosis in humans and animals.

[0011] These strains have been previously disclosed in WO2021129998.

[0012] In a preferred embodiment, the modulation of the composition and activity of the gut microbiota comprises: a) the preparation results in an increase in the taxa belonging to Bifidobacterium, Lactobacillus, Akkermansia muciniphila, Streptococcus and Faecalibacterium prausnitzii; b) the preparation results in a reduction of taxa belonging to the following genus Proteobacteria, Neisseria, Neisseria flavescens, Escherichia coli, Bordetella, Shigella, Salmonella, Bacteroides, Prevotella, Helicobacter pylori, Yersinia, Pseudomonas, Pseudomonas aeruginosa and Klebsiella; c) The preparation results in an increase in the alpha or beta diversity of the gut microbiota and an increase in the homogeneity of the gut microbiota One or more of the following is selected.

[0013] In a specific configuration, the preparation results in at least a 5% relative increase in the taxa belonging to Bifidobacterium, Lactobacillus, Akkermansia muciniphila, Streptococcus, and Faecalibacterium prausnitzii compared to a placebo group.

[0014] In another embodiment, the preparation provides at least a 5% relative reduction in the taxa belonging to Proteobacteria, Neisseria, Neisseria flavescens, Escherichia coli, Bordetella, Shigella, Salmonella, Bacteroides, Prevotella, Helicobacter pylori, Yersinia, Pseudomonas, Pseudomonas aeruginosa, and Klebsiella compared to a placebo group.

[0015] In another embodiment, the preparation results in at least a 5% relative increase in alpha or beta diversity of the gut microbiota and an increase in the homogeneity of the gut microbiota compared to a placebo group.

[0016] The cells of the strains of the invention can be present in the compositions of the invention as spores (dormant), as plant cells (growing), as transitional cells (transitioning from plant cells to spores or vice versa), as cell extracts, or as a combination of at least two of these types of cells. In a preferred embodiment, the probiotic strains are present in dormant form or as plant cells. In alternative embodiments, cytoplasmic extracts or cell-free supernatants or heat-killed biomasses of the probiotic strains are used.

[0017] In a further preferred embodiment, the preparation further comprises one or more probiotic strains, said strains being preferably selected from the genus Pediococcus, Weissella sp., more preferably Pediococcus pentosaceus DSM 33371.

[0018] In a further preferred embodiment the preparation is selected from the group consisting of: Aspergillus niger, Aspergillus oryzae, Bacillus sp., Lactobacillus sp., Pediococcus sp., Weissella sp., Rothia mucilaginosa, Rothia aeriana, aeria), subtilisins, nattokinase, arabinoxylan, barley cereal fiber, oat cereal fiber, rye fiber, wheat bran fiber, inulin, fructooligosaccharides (FOS), galactooligosaccharides (GOS), resistant starch, beta-glucan, glucomannan, galactoglucomannan, guar gum, xylooligosaccharides, and microbial proteases purified from alginates.

[0019] The present invention is also directed to the use of the preparation to correct a dysbiosis that typically occurs in the context of or preceding the onset of a gluten-related disorder, preferably selected from celiac disease, non-celiac gluten sensitivity, wheat allergy, and gluten-sensitive irritable bowel syndrome, in a subject or animal in need thereof.

[0020] In a preferred configuration, the preparation is for treating or preventing dysbiosis resulting from adherence to specific dietary practices, including gluten-free diets, foods with reduced intake of gluten or cereals or cereal-derived or cereal-containing foods.

[0021] Further, the preparation is for treating or preventing dysbiosis, preferably dysbiosis associated with type 2 diabetes, obesity, non-alcoholic fatty liver disease, allergic diseases, major depressive disorder, Parkinson's disease, Alzheimer's disease, autoimmune diseases.

[0022] In a preferred embodiment, the preparation further comprises a substance, preferably an alginate, that acts as a permeabilizer of the microbial cell membrane of a member of the genera Bacillus sp., Lactobacillus sp., Pediococcus sp., or Weissella sp.

[0023] In an alternative embodiment, one or more of the probiotic strains selected from Bacillus sp., Lactobacillus sp., Pediococcus sp., and Weissella sp. are immobilized individually or as a consortium. Immobilization can be achieved on a solid surface such as cellulose or chitosan, or by incorporation into a porous matrix such as polysaccharide gels such as alginate, k-carrageenan, agar, chitosan, polygalacturonic acid, or other polymer matrices such as gelatin, collagen, polyvinyl alcohol, by aggregation and microencapsulation, or by electrospray techniques.

[0024] A subject of the present invention is the use of the preparation according to the invention, which is a food or feed supplement, or a functional food or edible product, or a medicine.Preferred foodstuffs according to the present invention are chocolate products, gummies, muesli, muesli bars, and dairy products.

[0025] A further subject of the invention is also the use of the preparation according to the invention as a synbiotic ingredient in an edible product.

[0026] A further subject of the present invention is the use of the preparation as a food composition further comprising at least one further food ingredient, preferably selected from proteins, carbohydrates, fats, further probiotics, prebiotics, enzymes, vitamins, immunomodulators, milk substitutes, minerals, amino acids, coccidiostats, acid-based products, pharmaceuticals, and combinations thereof.

[0027] In a specific configuration, the preparation is formulated for oral use, preferably as a pill, capsule, tablet, granulated powder, operculum, soluble granules, sachets, pills or drinkable vials, or as a syrup or drink, or added to a food product, preferably cereal, gummies, bread, muesli, muesli bars, health bars, biscuits, chocolate, yogurt or spreads.

[0028] Food compositions according to the invention also include dietary supplements, for example in the form of pills, capsules, tablets, powders or liquids.

[0029] A further subject of the invention is the use of the preparation according to the invention as a pharmaceutical composition comprising the preparation and a pharma- ceutically acceptable carrier. [Brief description of the drawings]

[0030] [Figure 1] FIG. 1 shows an outline of a human gluten challenge test. [Diagram 2] FIG. 1 shows that daily consumption of a probiotic composition beneficially modulates the composition of the gut microbiota.

[0031] Working Example Example 1: Overview of gluten challenge test An example of the study setup is shown in Figure 1. Treatment groups received active or placebo capsules for 34 days, while following a gluten-free diet from days 1 to 41 and consuming prescribed amounts of gluten in capsule or bread form, stepwise from 50 mg to 10 g per day from day 11 onwards.

[0032] Figure 1 shows an overview of the human gluten challenge study. Healthy subjects aged 18–50 years were divided into two groups: (i) 40–50 received probiotics and (ii) 20–30 received placebo. From day 1 to day 41 of the study, subjects received capsules of probiotics or placebo. Both groups followed a gluten-free diet for the first 10 days, adhering to the elimination of residual traces of gluten and similar proteins from the feces. After 10 days, both groups started gluten administration as follows (see Figure 1): 50 mg / day (gluten capsule) for 4 days, 1 g / day (gluten capsule) for 4 days, 3 g / day (same amount of slide pan reintroduced) for 4 days, and 10 g / day (same amount of slide pan reintroduced) for 17 days. This final gluten dose corresponds to the average intake of gluten in most European countries. At this stage (10 days + 4 days + 4 days + 4 days + 10 days + 7 days = 41 days in total), the administration of the active and placebo preparations was stopped and a 7-day washout period was included. The washout period was included to provide information on the ability of the probiotic preparations to colonize the gastrointestinal tract for a longer period. The number of participants was calculated based on the statistical power estimates to be able to detect statistically different effects comparing the active and placebo groups. Fecal samples were collected at the beginning and end of each period for microbiological, glutenological, immunological, and metabolomic analyses.

[0033] Example 2: Effect of a probiotic formulation on gut microbiome composition, richness, and α / β diversity [Table 1] Table 1: Comparison of fecal microbiota composition, richness and alpha diversity of probiotic compositions (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) capsules and placebo capsules taken once daily in healthy adults. Active and placebo capsules were consumed on a background of gluten-controlled diets (gluten-free, 50 mg gluten per day, 1 g gluten per day, or 10 g gluten per day) as shown in Figure 1. "↑" represents a significant increase in the abundance of a taxon in the probiotic group compared to the placebo group, and "↓" the opposite. Exemplary parameters in the fields marked with asterisks are shown in detail in Figure 2.

[0034] Figure 2 shows that daily intake of a probiotic composition containing 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 beneficially modulates the composition of the gut microbiota. Observed OTU counts and richness indices (Chao1, Shannon, Simpson, and Fisher) between individuals treated with probiotic (n=33) or placebo (n=11). Pairwise Wilcoxon signed-rank tests were used to compare means between placebo and probiotic groups (p<0.05).

[0035] In addition to the data presented in Table 1, the abundance of Bacillus, Lacticaseibacillus, Lactiplantibacillus, Limosilactobacillus, Lacticaseibacillus paracasei, and Limosilactibacillus reuteri were all higher in subjects supplemented with probiotics compared to placebo. In conclusion, probiotic intake improved many microbial parameters reported to be impaired in gluten-related disorders and in the context of a gluten-free diet.

[0036] Analysis of fecal samples was performed with culture-dependent and culture-independent methods to estimate the viability of the administered probiotics and, more generally, their impact on the gastrointestinal microbiota. In the culture-dependent approach, selective media were used to quantify the viability of the administered probiotics. A mixture of fecal samples (5 g) and 45 ml of sterile physiological solution was homogenized. The relatively selective media were: - MRS agar and Rogosa agar for putative Lactobacillus; - LBG agar for presumptive Bacillus.

[0037] RAPD-PCR analysis and partial sequencing of the 16S gene were performed to identify the species / strains of the administered probiotic preparations obtained from faeces. Based on the genome sequences of the administered probiotics, specific probes were designed using RT-PCR analysis to confirm the identification 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 in the feces. For culture-independent analysis, RNA was extracted from approximately 200 mg aliquots of fecal samples using the Stool total RNA purification kit (Norgen Biotek Corp., Ontario, Canada, USA). The quality and abundance of the RNA extracts were measured using 1% agarose-0.5X TBE gels and spectrophotometric measurements at 260 nm, 280 nm, and 230 nm with a NanoDrop ND-1000 Spectrophotometer. A 1 μg aliquot of the extracted total RNA was transcribed into cDNA using random exomers and the Tetro cDNA synthesis kit from Bioline (Bioline USA Inc, Tanunton, MA, USA) according to the manufacturer's instructions. Primers: forward primer 28F:GAGTTTGATCNTGGCTCAG and reverse primer 519R:GTNTTACNGCGGCKGCTG, based on the V1-V3 region of the 16S rRNA gene (Escherichia coli positions 27-519), were used to detect the fecal microbiome. cDNA sequencing analysis was performed using the Illumina platform.Raw sequence data were screened, trimmed, and filtered using the QIIME pipeline version 1.4.0 (http: / / qiime.sourceforge.net) with default settings. Chimeras were removed using B2C2 (http: / / www.researchandtesting.com / B2C2.html). Sequences shorter than 250 bp were removed. Chimera-free FASTA sequences of each sample were evaluated using BLASTn against a database from GenBank (http: / / ncbi.nlm.nih.gov). Sequences were first clustered into OTU (Operational Taxonomic Unit) clusters with 97% identity (3% divergence) using USEARCH. To determine bacterial identity, sequences were first queried against a high-quality 16S bacterial sequence database from NCBI using a distributed BLASTn.NET algorithm. Database sequences were judged to be of high quality based on criteria originally described by the Ribosomal Database Project (RDP, v10.28). Indices of alpha diversity (indices of rarefaction, Good's coverage, Chao1 richness, Pielou's evenness, and Shannon diversity) and beta diversity were calculated and plotted using QIIME. The final dataset at species and other relevant taxonomic levels was compiled in a separate worksheet for compositional analysis between fecal samples and treatments.

[0038] Additionally, fecal samples were subjected to 16S rRNA gene amplification and sequencing as described

[24] : for sequencing of the microbial composition, all fasting samples were analyzed by 16S rRNA gene amplification and sequencing by Biomes NGS GmbH (Wildau, Germany). Microbial genomic DNA was extracted from feces by bead-beating. Using the most promising bacterial and archaeal primer pairs

[25] , the V3-V4 region of the 16S rRNA gene was amplified and sequenced using a 2 × 300 bp paired-end protocol on an Illumina MiSeq platform according to the manufacturer's instructions (Illumina, San Diego, CA, USA).

[0039] Bioinformatics Raw microbial sequences were processed using the QIIME (Quantitative Insights Into Microbial Ecology) pipeline

[26] . High-quality reads were binned into OTUs (Operational Taxonomic Units) at a similarity threshold of 97% using UCLUST

[27] and a “de novo” approach. Taxonomic assignment was performed using the Ribosomal Database Project (RDP) classifier against the Greengenes database. All singleton OTUs were removed to remove the majority of chimeric sequences. Microbial α-diversity was analyzed using the Chao1 index, Shannon entropy, Simpson index, and phylogenetic diversity whole-tree metrics, and β-diversity was estimated based on the Bray-Curtis dissimilarity index and plotted as multidimensional scaling or principal coordinate analysis (PCoA) using CLC Genomics Workbench version 20.0.4 (QIAGEN). The mean differences in α-diversity indices were analyzed by GraphPad Prism version 9.0.0 (San Diego, CA, USA) using the Mann-Whitney U test and plotted as mean ± SD. A p-value < 0.05 was considered statistically significant. Differences in microbial community composition (β-diversity) between each group were tested using permutation multivariate analysis of variance (PERMANOVA).

[0040] Example 3: Effect of probiotic preparations on gut microbiome activity To explore changes in the functional capacity of the gut microbiome between the active treatment groups (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) and placebo groups and in response to dietary gluten intake, fecal 16S sequence data of study participants were analyzed as described above. Based on the sequence profiles of marker genes, the functional potential of the bacterial community was predicted using PICRUSt 2

[28] , and the results were compared between the active and placebo groups for each dietary gluten intake regime. As shown in Table 2, probiotic intake improved the protein degradation and carbohydrate metabolism capabilities of the gut microbiome, regardless of the amount of gluten intake.

[0041] [Table 2] Table 2: Comparison of PICRUSt2 predicted metabolic functions of fecal 16S rRNA microbiome data from healthy adults receiving one capsule per day of probiotic compositions (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) capsules and a placebo capsule. Active and placebo capsules were taken on a background of gluten-controlled diets (gluten-free, or 50 mg gluten per day, or 1 g gluten per day), as shown in Figure 1. "↑" represents a significant increase in the abundance of predicted metabolic functions in the probiotic group compared to the placebo group, and "↓" the opposite.

[0042] literature [Table 3-1] [Table 3-2]

Claims

1. 1. Use of a preparation for treating or preventing dysbiosis in humans and animals, said preparation being selected from the group consisting of Lactobacillus plantarum DSM 33363, Lactobacillus plantarum DSM 33364, Lactobacillus paracasei DSM 33373, Lactobacillus reuteri DSM 33374, Bacillus megaterium DSM 33300, Bacillus pumilus DSM 33301, Bacillus pumilus DSM 33302, Bacillus pumilus DSM 33303, Bacillus pumilus DSM 33304, Bacillus pumilus DSM 33305, Bacillus pumilus DSM 33306, Bacillus pumilus DSM 33307, Bacillus pumilus DSM 33308, Bacillus pumilus DSM 33309, Bacillus pumilus DSM 33310, Bacillus pumilus DSM 33311, Bacillus pumilus DSM 33312, Bacillus pumilus DSM 33313, Bacillus pumilus DSM 33314, Bacillus pumilus DSM 33315, Bacillus pumilus DSM 33316, Bacillus pumilus DSM 33317, Bacillus pumilus DSM 33318, Bacillus pumilus DSM 33319 ... Uses including Bacillus pumilus DSM 33297, and Bacillus pumilus DSM 33355.

2. The preparation is used to modulate the composition and activity of the intestinal microbiota, the modulation being: a) the preparation results in an increase in the taxa belonging to the genus Bifidobacterium, Lactobacillus, Akkermansia muciniphila, Streptococcus, Faecalibacterium prausnitzii, b) the preparation is selected from the group consisting of Proteobacteria, Neisseria, Neisseria flavescens, Escherichia coli, Bordetella, Shigella, Salmonella, Bacteroides, Prevotella, Helicobacter pylori, Yersinia, Pseudomonas, Pseudomonas aeruginosa, aeruginosa), resulting in a decline in taxa belonging to Klebsiella, c) the preparation results in an increase in the alpha or beta diversity of the gut microbiota. The use according to claim 1, wherein the compound is selected from one or more of the following:

3. The use according to claim 1, wherein the consortium of strains results in an increase in the proteolytic and carbohydrate metabolic capacity of the intestinal microbiome of humans and animals.

4. 4. Use according to any one of claims 1 to 3, wherein the probiotic strain is present in a dormant form or as plant cells.

5. 4. The use according to any one of claims 1 to 3, wherein the preparation further comprises one or more probiotic strains.

6. The preparation is selected from the group consisting of Aspergillus niger, Aspergillus oryzae, Bacillus sp., Lactobacillus sp., Pediococcus sp., Weissella sp., Rothia mucilaginosa, Rothia aeriana, and the like.

4. The use according to any one of claims 1 to 3, further comprising one or more of the following microbial proteases purified from maltodextrin, ...

7. 4. The use according to any one of claims 1 to 3, wherein the preparation is for treating or preventing a gluten-related disorder in a subject or animal in need thereof.

8. 4. Use according to any one of claims 1 to 3 for treating or preventing dysbiosis resulting from adherence to special dietary practices, including gluten-free diets, foods with reduced intake of gluten or cereals or cereal-derived or cereal-containing foods.

9. 4. Use according to any one of claims 1 to 3 for treating or preventing dysbiosis.

10. 4. The use according to any one of claims 1 to 3, further comprising a substance that acts as a permeabilizer for the microbial cell membrane of a member of the genera Bacillus, Lactobacillus, Pediococcus, or Weissella.

11. 4. The use according to any one of claims 1 to 3, wherein one or more of the probiotic strains selected from the genera Bacillus, Lactobacillus, Pediococcus, and Weissella are immobilized individually or as a consortium.

12. 4. The use according to any one of claims 1 to 3, wherein the preparation is a food or feed supplement, or a functional food or edible product, or a pharmaceutical product.

13. 13. The use according to claim 12, wherein the preparation is formulated for oral administration or as a syrup or drink or added to food.

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