SYNBIOTIC COMPOSITIONS

Abstract

A synbiotic preparation may include at least one probiotic strain and at least one amino acid or derivative thereof selected from glutamine, glutamic acid or salts thereof, conjugated glutamine, or oligopeptides of 2-10 amino acid units in length, wherein the amino acid units may be natural amino acids, and at least one amino acid unit being a glutamine or glutamic acid unit.

Claims

1. A preparation, comprising: a probiotic strain comprising Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens and/or Bacillus pumilus; and an amino acid comprising glutamine, a salt of glutamine, glutamic acid, a salt of glutamic acid, conjugated glutamine, and/or an oligopeptide of 2-10 natural amino acid units in length, the oligopeptide comprising a glutamine or glutamic acid unit.

2. The preparation of claim 1, wherein the probiotic strain is Bacillus subtilis DSM 32315, Bacillus subtilis DSM 32540, Bacillus licheniformis DSM 32314, Bacillus amyloliquefaciens CECT 5940, Bacillus subtilis DSM 32592, and/or Bacillus pumilus DSM 32539.

3. The preparation of claim 1, wherein the oligopeptide is present an further comprises an alanine or glycine unit.

4. The preparation of claim 1, wherein the oligopeptide is present and is a dipeptide, and wherein the dipeptide comprises glycine-glutamine, glycine-glutamic acid, alanine-glutamine, and/or alanine-glutamic acid, the dipeptide being optionally acetylated.

5. The preparation of claim 1, wherein a total amount of probiotic strain and amino acid or oligopeptide is at least 40 wt. % of total preparation weight.

6. The preparation of claim 1, wherein the preparation comprises an enteric coating comprising a methyl acrylate-methacrylic acid copolymer, cellulose acetate phthalate, cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate, polyvinyl acetate phthalate, methyl methacrylate-methacrylic acid copolymer, shellac, cellulose acetate trimellitate, sodium alginate, and/or zein.

7. A feed or food supplement, comprising the preparation of claim 1.

8. A synbiotic ingredient suitable for feed or a food product, comprising the preparation of claim 1.

9. A feed or foodstuff composition, comprising: the preparation of claim 1; and a further feed or food ingredient optionally comprising a protein, carbohydrate, fat, further probiotic, prebiotic, enzyme, vitamin, immune modulator, milk replacer, mineral, amino acid, coccidiostat, acid-based product, and/or medicine.

10. A pharmaceutical composition, comprising: the preparation of claim 1; and a pharmaceutically acceptable carrier.

11. The composition of claim 9, suitable to prevent or treat diarrhea, constipation, irritable bowel syndrome, Crohn's disease, ulcerative colitis, colorectal cancer, bowel cancer, cardiovascular disease, arteriosclerosis, fatty liver disease, hyperlipidemia, hypercholesterolemia, obesity, adipositas, type 2 diabetes, metabolic syndrome, chronic inflammatory disease, or allergic disease.

12. A capsule comprising: a probiotic strain comprising Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, and/or Bacillus pumilus, and a dipeptide comprises glycine-glutamine, glycine-glutamic acid, alanine-glutamine, and/or alanine-glutamic acid, the dipeptide being optionally acetylated.

13. The capsule of claim 12, comprises between 1×10.sup.8 and 2×10.sup.10 CFU of the probiotic strain and between 50 mg and 800 mg of the dipeptide.

14. The capsule of claim 12, wherein the probiotic strain and dipeptide are present in an amount of at least 40 wt. % of total capsule filling weight.

15. The capsule of claim 12, further comprising: vitamin A as all-trans-retinol, all-trans-retinyl-ester(s), all trans-beta-carotene, and/or other provitamin A carotenoid(s); vitamin B1 as thiamine; vitamin B2 as riboflavin; vitamin B3 as niacin; vitamin B5 as pantothenic acid; vitamin B6 as pyridoxine; vitamin B7 as biotin; vitamin B9 as folic acid or folate; vitamin B12 as cobalamin(s); vitamin C as ascorbic acid; vitamin D as calciferol(s); vitamin E as tocopherol(s) and/or tocotrienol(s); and/or vitamin K as quinone(s).

16. The capsule of claim 12, further comprising; sulfur, iron, chlorine, calcium, chromium, cobalt, copper, zinc, magnesium, manganese, molybdenum, iodine, and/or selenium.

17. The capsule of claim 12, further comprising: an inulin, fructooligosaccharide, galactooligosaccharide, starch, pectin, beta-glucan, and/or xylooligosaccharide.

18. The capsule of claim 12, further comprising: a plant extract, optionally from broccoli, olive fruit, pomegranate, blackcurrant, blueberry, bilberry, sea buckthorn, camu camu, boysenberry, curcuma, ginger, garlic, grape seeds, acai berry, aronia, goji berry, horseradish, boswellia serrata, spirulina, panax ginseng, cannabidiol, rose hip, pu erh, sencha, echinacea, and/or green tea leaves.

19. The capsule of claim 12 further comprising: an enteric coating comprising a methyl acrylate-methacrylic acid copolymer, cellulose acetate phthalate, cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate, polyvinyl acetate phthalate, methyl methacrylate-methacrylic acid copolymer, shellac, cellulose acetate trimellitate, sodium alginate, and/or zein.

Description

WORKING EXAMPLES

Example 1: strains Bacillus subtilis DSM 32315, Bacillus subtilis DSM 32540, Bacillus licheniformis DSM 32314 and Bacillus amyloliquefaciens CECT 5940 are able to persist in the colonic human microbiota

[0083] Intestinal screening model

[0084] To determine the effect of the probiotic strains Bacillus subtilis DSM 32315, Bacillus subtilis DSM 32540, Bacillus licheniformis DSM 32314 and Bacillus amyloliquefaciens CECT 5940 on adult colonic microbiota, an intestinal screening model was used (i-screen, TNO, the Netherlands). Therefore the i-screen model was inoculated with standard human adult fecal microbiota material, which consisted of pooled fecal donations from 6 healthy adult volunteers (Caucasian, European lifestyle and nutrition). The fecal material was mixed and grown in a fed-batch fermenter for 40 hours to create a standardized microbiota as described previously [21]. These standard adult gut microbiota sets were stored at −80° C. in 12% glycerol.

[0085] The intestinal microbiota was cultured in vitro in modified standard ileal efflux medium (SIEM), the composition of which was described by [22] and modified as follows: 0.047 g/l pectin, 0.047 g/l xylan, 0.047 g/l arabinogalactan, 0.047 g/l amylopectin, 0.392 g/l starch, 24.0 g/l casein, 24.0 Bacto pepton, 0.4 ox-bile and 0.2 g/l cysteine. All components were supplied by Trititium Microbiology (Veldhoven, The Netherlands). The pH of the medium was adjusted to 5.8.

[0086] For the i-screen fermentations, the precultured standardized fecal inoculum was diluted 50 times in 1350 μl modified SIEM. All experiments have been carried out in triplicates. The strains Bacillus subtilis (DSM 32315), Bacillus subtilis DSM 32540, Bacillus licheniformis DSM 32314 and Bacillus amyloliquefaciens CECT 5940 were precultured separately in 50 ml LBKelly medium [23], for about 16 h. Incubation was done in shaking flasks at 37° C. under aerobic conditions. After incubation, bacterial density was determined by optical density measurement at 600 nm. A final stock solution of 1×10.sup.10 cells/ml was prepared in 1 ml buffer solution (0.1 mM MES pH 6). The suspension of each strain was introduced into the i-screen to a final level of about 10.sup.9 cells/ml, respectively

[0087] The i-screen incubation was performed under following gas conditions: 0.2% O.sub.2, 0.2% CO.sub.2, 10% H.sub.2, 89.6% N.sub.2.

DNA isolation

[0088] DNA extraction for the sequencing of 16S rRNA coding genes was performed as described by Ladirat et al. (2013) with some minor modifications. Approximately 100 μl of the culture materials were added to the wells of a 96 well plate containing per well 300 μl of lysis buffer (Mag Mini DNA Isolation Kit, LGC ltd, UK), 500 μl zirconium beads (0.1 mm; BioSpec products, Bartlesville, Okla., USA) and 500 μl of phenol saturated with Tris-HCI (pH 8.0; Carl Roth GMBH, Germany). The 96 well plate was placed in a Mini-BeadBeater-96 (BioSpec products, Bartlesville, Okla, USA) for 2 min at 2100 oscillations/min. DNA was subsequently purified using the Agowa Mag Mini DNA Isolation Kit according to the manufacturer recommendations. Extracted DNA was eluted in a final volume of 60 μl buffer.

V4 16S rRNA gene sequencing

[0089] The microbiota composition was analyzed by 16S rRNA gene amplicon sequencing of the V4 hypervariable region. This was achieved through a series of steps:

[0090] The amount of bacterial DNA in the i-screen DNA samples was determined by quantitative polymerase chain reaction (qPCR) using primers specific for the bacterial 16S rRNA gene: Forward primer: CGAAAGCGTGGGGAGCAAA; SEQ ID NO. 1; Reverse primer: GTTCGTACTCCCCAGGCGG SEQ ID NO. 2: ; Probe: 6FAM-ATTAGATACCCTGGTAGTCCA-MGB SEQ ID NO. 3.

[0091] Subsequently, PCR amplicons of the V4 hypervariable region of the 16S rRNA gene were generated for the individual samples by amplification of 500 μg of DNA as described by [24] (2013), using F515/R806 primers [25]. Primers included Illumina adapters and a unique 8-nt sample index sequence key [24]. A mock control was included for technical quality control. The amount of amplified DNA per sample was quantified using the dsDNA 910 Reagent Kit on the Fragment Analyzer (Advanced Analytical). The amplicon libraries were pooled in equimolar amounts and purified from 1,2% agarose gel using the Gel Extraction Kit (Qiagen). The Library was quantified using the Quant-iT™ PicoGreen® dsDNA Assay Kit (Thermo Fisher Scientific). Paired-end sequencing of amplicons was conducted on the Illumina MiSeq platform (Illumina, Eindhoven, The Netherlands).

[0092] The sequence data was processed with Mothur v.1.36.1 [26] in line with the mothur MiSeq SOP [24]. Before merging the read pairs, low quality regions were trimmed using Btrim [27] with a sliding window size of 5 nt and average quality score of 25. After merging, the sequences were filtered by length while no ambiguous bases were allowed. The unique sequences were aligned to the bacterial SILVA SEED reference alignment release 102 (available at: http://www.mothur.org/wiki/Silva_reference_files); too short sequences were removed using screen. seqs with parameters “optimize=start-end, criteria=90”. Chimeric sequences were identified per sample using UCHIME [28] in de novo mode and removed. Next, sequences occurring less than 10 times in the entire dataset were removed. Taxonomic names were assigned to all sequences using the Ribosomal Database Project (RDP) naïBayesian classifier with confidence threshold of 60% and 1000 iterations [29] and the mothur-formatted version of the RDP training set v.9 (trainset9_032012).

[0093] Sequences were grouped using Minimum Entropy Decomposition (MED) algorithm that clusters 16S rRNA gene amplicons in a sensitive manner [30]. To filter noise, the “minimum substantive abundance” filter was set to 200.

[0094] The colonic human microbiota in the i-screen was also supplemented with viable vegetative Bacillus spp. cells of each probiotic strain.

[0095] Based on MiSeq sequencing of the V4 hypervariable region of the 16S rRNA encoding gene specific effects on the microbiota composition related to the individual strains could be visualized.

[0096] Based on the number of sequence reads, the abundance of bacilli was high at start of the i-screen experiment (about 10.sup.9 cfu/ml), and thus Bacillus subtilis DSM 32315 contributed to approximately 91% of the total bacterial population at t=0 h (FIG. 1 C), Bacillus subtilis DSM 32540 contributed to approximately 87% of the total bacterial population at t=0 h (FIG. 1 E), Bacillus licheniformis DSM 32314 contributed to approximately 94% of the total bacterial population at t=0 h (FIG. 1 G) and Bacillus amyloliquefaciens CECT 5940 contributed to approximately 88% of the total bacterial population at t=0 h (FIG. 1 I).

[0097] The control without added Bacillus spp. strain is shown at time point 0 h (FIG. 1 A) and after 24 h of incubation (FIG. 1 B). Upon 24 h incubation, the fecal microbiota was able to recover on the expense of the relative presence of the bacilli. It appeared that the DSM 32315 cells were persistent being present at a level of 41% in i-screen after 24 h incubation (FIG. 1 D), the DSM 32540 cells were persistent being present at a level of 19% in i-screen after 24 h incubation (FIG. 1 F), the DSM 32314 cells were persistent being present at a level of 4% in i-screen after 24 h incubation (FIG. 1 H) and the CECT 5940 cells were persistent being present at a level of 7% in i-screen after 24 h incubation (FIG. 1 J).

[0098] FIG. 1 shows pie Charts showing at genus level of i-screen fermentation samples based on MiSeq sequencing of the V4 hypervariable region of the 16S rRNA encoding region gene. The detected genera and their relative abundance are represented by shaded sections. A) 0 h incubation without any addition B) after 24 h incubation without any addition C) 0 h after addition of vegetative Bacillus subtilis DSM 32315 cells D) 24 h after addition of vegetative Bacillus subtilis DSM 32315 cells E) 0 h after addition of vegetative Bacillus subtilis DSM 32540 cells F) 24 h after addition of vegetative Bacillus subtilis DSM 32540 cells G) 0 h after addition of vegetative Bacillus licheniformis DSM 32314 cells H) 24 h after addition of vegetative Bacillus licheniformis DSM 32314 cells I) 0 h after addition of vegetative Bacillus amyloliquefaciens CECT 5940 cells J) 24 h after addition of vegetative Bacillus amyloliquefaciens CECT 5940 cells.

[0099] Example 2: Probiotic strains Bacillus subtilis DSM 32315, Bacillus subtilis DSM 32540, Bacillus licheniformis DSM 32314 and Bacillus amyloliquefaciens CECT 5940 produce significant levels of lactate

[0100] In the SIEM at start, a low level of lactate was detected at about 0.04 mg/ml, which fully disappeared in the presence of microbiota with a content of <0.02 mg/ml lactate after 24 h (FIG. 2). The Bacillus strains produced significant levels of lactate in the SIEM without colon community after 24 h. The cells of DSM 32315 produced 0.35 mg/ml, the cells of DSM 32540 produced 0.31 mg/ml, the cells of DSM 32314 produced 0.19 mg/ml, and the cells of CECT 5940 produced 0.11 mg/ml.

[0101] After 24 h incubation with human gut microbiota the amount of lactate was still significantly increased to 0.12 mg/ml in the presence of DSM 32315 or DSM 32540 cells and to 0.15 g/ml in the presence of DSM 32314, respectively.

[0102] Thus, these probiotic strains give rise to a significant lactate formation by the human gut microbiota. This can be interpreted as a beneficial effect, because lactate can be transformed into health promoting SCFAs.

[0103] FIG. 2 shows after 24 h incubation in SIEM measured lactate concentrations in mg/ml in SIEM and in the presence of colon microbiota containing Bacillus subtilis DSM 32315, Bacillus subtilis DSM 32540, Bacillus licheniformis DSM 32314 and Bacillus amyloliquefaciens CECT 5940, respectively. Limit detection of lactate was 0.02 mg/ml.

Example 3: Probiotic strains Bacillus subtilis DSM 32315, Bacillus licheniformis DSM 32314 and Bacillus amyloliquefaciens CECT 5940 support acetate production by the human microbiota community, respectively

[0104] The Bacillus strains DSM 32315, DSM 32314 and CECT 5940 cause a substantial increase in the acetate production of the gut microbiota. FIG. 3 shows an average acetate level of about 44 mM in the i-screen after 24 h exposure without added probiotic strains, while acetate is with 3.4 mM in the SIEM hardly detectable.

[0105] After 24 h incubation increases of 10.4 mM, 43.7 mM and 3.3 mM in acetate concentrations are observed in the presence of the strains DSM 32315, DSM 32314 and CECT 5940, respectively (FIG. 3).

[0106] The p-value for all data are below 0.05, which means they are statistically significant. Thus, the probiotic strains DSM 32315, DSM 32314 and CECT 5940 supports acetate production, which has a beneficial effect on the human gut.

[0107] FIG. 3 shows after 24 h incubation in SIEM measured acetate concentrations in mM in SIEM and in the presence of colon microbiota containing Bacillus subtilis DSM 32315, Bacillus licheniformis DSM 32314 and Bacillus amyloliquefaciens CECT 5940, respectively.

Example 4: Probiotic strains Bacillus subtilis DSM 32315, Bacillus subtilis DSM 32540, Bacillus licheniformis DSM 32314 and Bacillus amyloliquefaciens CECT 5940 support the production of propionate by the human microbiota, respectively

[0108] The Bacillus strains DSM 32315, DSM 32540, DSM 32314 and CECT 5940 do not produce propionate in SIEM, but they significantly (p-value <0.05) accelerate propionate production by human microbiota (FIG. 4). The strains support significantly the production of propionate by the gut microbiota compared to the control. FIG. 4 shows an average propionate level of about 11.5 mM in the i-screen after 24 h exposure without added probiotic strains, while propionate is with 2.7 mM hardly detectable in the SIEM. In the presence of vegetative Bacillus subtilis DSM 32315 cells the amount of propionate was 15.1 mM higher than in the control after 24 h. In the presence of vegetative Bacillus subtilis DSM 32540 the propionate amount was 12.2 mM higher. In the presence of DSM 32314 the amount was 13.4 mM higher, and in the presence of CECT 5940 the amount of propionate was 7.1 mM higher.

[0109] Propionate is beneficial for the health status of the human gut, because it can be incorporated into gluconeogenesis.

[0110] FIG. 4 shows after 24 h incubation in SIEM measured propionate concentrations in mM in SIEM and in the presence of colon microbiota containing Bacillus subtilis DSM 32315, Bacillus subtilis DSM 32540, Bacillus licheniformis DSM 32314 and Bacillus amyloliquefaciens CECT 5940, respectively.

Example 5: Probiotic strains Bacillus subtilis DSM 32315, Bacillus licheniformis DSM 32314 and Bacillus amyloliquefaciens CECT 5940 support the production of n-butyrate in a human microbiota composition, respectively

[0111] The probiotic strains Bacillus subtilis DSM 32315, Bacillus licheniformis DSM 32314 and Bacillus amyloliquefaciens CECT 5940 do not produce detectable levels of n-butyrate after exposure in SIEM for 24 h, but they have significant positive influences (p-values <0.05) on the level of n-butyrate production by the human microbiota (FIG. 5). FIG. 5 shows an average n-butyrate level of about 5.4 mM in the i-screen after 24 h exposure without added probiotic strains, while propionate is hardly detectable in the SIEM with 0.3 mM. In the presence of vegetative Bacillus subtilis DSM 32315 cells the amount of n-butyrate was 2.0 mM higher than in the control after 24 h. In the presence of vegetative cells of DSM 32314 the n-butyrate amount was 1.0 mM higher, and in the presence of vegetative cells of CECT 5940 the n-butyrate amount was 4.8 mM higher. The generation of higher n-butyrate amounts can be beneficial for the lipid biosynthesis, e.g. the production gut hormones.

[0112] FIG. 5 shows after 24 h incubation in SIEM measured n-butyrate concentrations in mM in the presence of colon microbiota containing Bacillus subtilis DSM 32315, Bacillus licheniformis DSM 32314 and Bacillus amyloliquefaciens CECT 5940, respectively.

Example 6: The strains Bacillus subtilis DSM 32315 and DSM 32540 reduce the iso-butyrate and iso-valerate formation in a human microbiota composition, respectively

[0113] FIG. 6 shows an average iso-butyrate level of about 0.73 mM and an average iso-valerate level of about 2.2 mM in the i-screen after 24 h exposure without probiotic strains added, while iso-butyrate was not detectable and the content of iso-valerate was 0.24 mM in the SIEM.

[0114] In particular, the strains DSM 32315 and DSM 32540 significantly reduce (p-value <0.05) the iso-butyrate and iso-valerate production by the human gut microbiota. After 24 h incubation, the iso-butyrate content was 0.5 mM lower in the presence of cells of DSM 32315 and 0.4 mM lower in the presence of cells of DSM 32540 compared to the control, respectively.

[0115] The Bacillus strains DSM 32315 and DSM 32540 also have negative influence on the level of iso-valerate production when added to the human microbiota. The concentration is significantly (p-value <0.05) decreased in the i-screen by a value of 0.7 mM and 0.8 mM compared to the control, respectively.

[0116] Upon introduction of the Bacillus strains DSM 32315 in the gut microbiota in the i-screen the cells support production of n-butyrate and inhibit the formation of iso-butyrate and iso-valerate. This can be an indication of lowered protein fermentation process in the gut, which also indicates a reduced production of harmful by-products.

[0117] FIG. 6 shows after 24 h incubation in SIEM measured iso-butyrate, and iso-valerate concentrations in mM in the presence of colon microbiota containing Bacillus subtilis DSM 32315, and Bacillus subtilis DSM 32540, respectively.

[0118] Example 7: Presence of Bacillus subtilis DSM 32315, Bacillus subtilis DSM 32540, Bacillus licheniformis DSM 32314 and Bacillus amyloliouefaciens CECT 5940 reduce abundance of Clostridium XI cluster, respectively. The strains DSM 32315, DSM 32314 and CECT 5940 increase abundance of Clostridium XIVa cluster, respectively

[0119] When compared to the control at starting time point 0 h (FIG. 1) and the development of the microbiota after 24 h, the presence of strains DSM 32315, DSM 32540, DSM 32314 and CECT 5940 reduces the Clostridium XI cluster from 6% down to values below 3% of the total microbial community (FIG. 7). The Clostridium XIVa cluster was increased after 24 h from 5% in the control up to at least 8% of the total community in the presence of the probiotic cells, respectively. The i-screen incubation was performed under following gas conditions: 0.2% O.sub.2, 0.2% CO.sub.2, 10% H.sub.2, 89.6% N.sub.2.

[0120] Thus, in the presence of these probiotic strains the human microbiota composition is shifted to a more healthy community.

[0121] FIG. 7 shows bar graph showing at genus level of i-screen fermentation samples based on MiSeq sequencing of the V4 hypervariable region of the 16S rRNA encoding region gene. Shaded bars represent the detected genera Clostridium XI and Clostridium XIVa and their relative abundance.

Example 8: Addition of glutamine or derivates thereof changes the human microbial community significantly

[0122] The presence of glutamine- or glutamic acid-containing dipeptides influences the microbial community by a shift to an eradicated Clostridium group XI and an increased Clostridium group XIVa (FIG. 8). The single amino acids were added to a concentration of 3.5 mM and the dipeptides to a concentration of 7 mM. The i-screen exposure was performed as described in example 1 under following gas conditions: 0% O.sub.2, 0.2% CO.sub.2, 10% Hz, 90% N.sub.2. Moreover, Ala-Gln and Gly-Glu reduced the Clostridia XI group almost completely, which is linked to strong beneficial effects on gut health. As controls the dipeptide Gly-Tyr not containing any glutamine or glutamic acid, and the single amino acids glutamine (Gln) and glutamate (Glu) were tested, which had no positive effect on the microbial community.

[0123] FIG. 8 shows bar graph showing at genus level of i-screen fermentation samples based on MiSeq sequencing of the V4 hypervariable region of the 16S rRNA encoding region gene. Shaded bars represent the detected genera Clostridium XI and Clostridium XIVa and their relative abundance.

Example 9: The combined addition of Bacillus subtilis (DSM 32315), Bacillus licheniformis (DSM 32314) or Bacillus amyloliouefaciens (CECT 5940) with glutamine or derivates thereof show synergistic effects on changes microbial community and their short chain acid production than the single components

[0124] The effect of the combination of different probiotic strains together with several glutamine- and glutamic acid containing peptides was analyzed in detail by the method described in example 7. As controls, the effects of glutamine (Gln), glutamic acid (Glu), and the non-Glu/non-Gln dipeptide glycine-tyrosine (Gly-Tyr) alone and in combination with the probiotic strains were analyzed. The single amino acids were added to a concentration of 3.5 mM and the dipeptides to a concentration of 7 mM.

[0125] The microbiota composition was analyzed after 24 h incubation. Synergistic positive effects of several combinations on the microbial community composition were observerd, as revealed by a higher increase of percentage of the Clostridium XIVa group and a higher decrease of the Clostridium group XI compared to the single addition of amino acid or dipeptide and probiotic cells. This synergistic effect was observed for the combination of B. subtilis (DSM 32315) with Ala-Gln, Gly-Glu, and Gly-Gln, for B. licheniformis (DSM 32314) with Ala-Gln, Gly-Glu, and Gly-Gln, and for B. amyloliquefaciens (CECT 5940) with Ala-Gln, Gln, Gly-Glu, and Gly-Gln (table 1).

[0126] Thus, in the presence of these probiotic strains in combination with glutamine- or glutamic acid containing dipeptides the human microbiota composition is shifted to a more healthy community.

TABLE-US-00001 TABLE 1 After 24 h incubation of human microbiota in SIEM with added amino acids, dipeptides and, or probiotic cells (B. subillis DSM 32315, B. licheniformis DSM 32314, or B. amyloliquefaciens CECT 5940) detected genera Clostridium XI, Clostridium XIVa, and others and their relative abundance. added substrate control Gly-Tyr Gln Glyn-Gln Ala-Gln Ac-Ala-Gln Glu Gly-Glu microbiota Clostridium_XI 30% 31% 28%  5%  0% 27% 27%  0% Clostridium_XIVa  1%  1%  1%  4%  2%  1%  1%  4% other 69% 68% 72% 91% 98% 72% 72% 95% microbiota + DSM 32315 Clostridium_XI 30% 33% 24%  4%  0% 31% 25%  0% Clostridium_XIVa  2%  1%  2%  6%  4%  2%  3%  8% other 68% 66% 74% 90% 96% 67% 72% 92% microbiota + CECT 5940 Clostridium_XI 23% 28% 20%  2%  0% 21% 26%  0% Clostridium_XIVa  1%  1%  1% 11% 10%  1%  1%  9% other 76% 67% 79% 88% 90% 78% 73% 91% microbiota + CECT 5940 Clostridium_XI 26% 28% 21%  4%  0% 20% 18%  0% Clostridium_XIVa  1%  3%  9% 15%  6%  4%  1% 10% other 72% 70% 70% 81% 94% 76% 80% 90%

[0127] Example 10: The combined addition of B. subtilis (DSM 32315), B. licheniformis (DSM 32314) or B. amyloliquefaciens (CECT 5940) with glutamine, or glutamine- or glutamic acid-containing dipeptides show synergistic effects on the n-butyrate production of the microbial community

[0128] The effect of the combination of different probiotic strains together with glutamine, or glutamine- or glutamic acid-containing dipeptides containing peptides on the n-butyrate production of the microbial community was analyzed for the strain B. subtilis (DSM 32315) as described in example 1 and 5. The single amino acids were added to a concentration of 3.5 mM and the dipeptides to a concentration of 7 mM.

[0129] The n-butyrate content was analyzed after 24 h incubation. Synergistic positive effects on the n-butyrate concentration were observed for the combinations of B. subtilis (DSM 32315) with Gln, Gly-Gln, Ala-Gln, Glu, or Gly-Glu, (FIG. 9). This shows that this probiotic strain in combination with glutamine, or glutamic acid, or glutamine-containing peptides have an accelerating effect on the beneficial n-butyrate production of the microbial community. As a negative control the dipeptide Gly-Tyr not containing any glutamine or glutamic acid was tested, which had no positive effect on the n-butyrate production.

[0130] FIG. 9 shows after 24 h incubation in SIEM with human microbiota measured n-butyrate concentrations in mM in the presence of colon microbiota containing different amino acids, or dipeptides with and without the combination of B. subtilis DSM 32315 cells, respectively.

[0131] Example 11: The combined addition of B. licheniformis (DSM 32314) or B. amyloliquefaciens (CECT 5940) cells with glutamine, glutamine- or glutamic acid-containing dipeptides show synergistic effects on the n-butyrate production of the microbial community

[0132] The effects of the combination of different probiotic strains together with glutamine, or glutamine- or glutamic acid-containing dipeptides containing peptides on the n-butyrate production of the microbial community were analyzed for the strains B. licheniformis (DSM 32314) and B. amyloliquefaciens (CECT 5940) as described in example 1, 5, and 10. The single amino acids were added to a concentration of 3.5 mM and the dipeptides to a concentration of 7 mM.

[0133] The n-butyrate content was analyzed after 24 h incubation. Synergistic positive effects on the n-butyrate concentration were observed for the combinations of B. licheniformis (DSM 32314) with Gly-Gln, Ala-Gln, and Gly-Glu (FIG. 10). As well as for the combinations of B. amyloliquefaciens (CECT 5940) with Gln, and Gly-Gln.

[0134] This shows that this probiotic strain in combination with glutamine, or glutamic acid, or glutamine-containing peptides have an accelerating effect on the beneficial n-butyrate production of the microbial community. As a negative control the dipeptide Gly-Tyr not containing any glutamine or glutamic acid was tested, which had no positive effect on the n-butyrate production.

[0135] FIG. 10 shows after 24 h incubation in SIEM with human microbiota measured n-butyrate concentrations in mM in the presence of colon microbiota containing different amino acids, or dipeptides with and without the combination of B. licheniformis (DSM 32314) or B. amyloliquefaciens (CECT 5940) cells, respectively.

[0136] Example 12: Capsules comprising a Bacillus subtilis and dipeptides as a prebiotic

[0137] The following components were filled in HPMC capsules (size 3).

TABLE-US-00002 TABLE 2 Preparations for filling into HPMC capsules Compound Capsule I Capsule II Capsule III Bacillus subtilis 66 mg (2 × 10.sup.9 CFU) 10 mg (1 × 10.sup.8 CFU) 300 mg (2 × 10.sup.10 CFU) B21 Dipeptide Ala-Gin 400 mg 50 mg 800 mg Vitamin B12 0.0125 mg 0.0125 mg 0.0125 mg Vitamin B6 0.7 mg 0.7 mg 0.7 mg Zinc 5 mg 5 mg 5 mg Biotin 25 mg 25 mg 25 mg

[0138] The capsules may further contain further prebiotic ingredients, selected from inulins, fructooligosaccharides (FOS), galactooligosaccharides (GOS), starch, pectin, beta-glucans and xylooligosaccharides.

[0139] The capsules may further contain one or more plant extracts, selected from broccoli, olive fruit, pomegranate, blackcurrant, blueberry, bilberry, sea buckthorn, camu camu, boysenberry, curcuma, ginger, garlic, grape seeds, acai berry, aronia, goji berry, horseradish, boswellia serrata, spirulina, panax ginseng, cannabidiol, rose hip, pu erh, sencha, echinacea, green tea leaves.

[0140] The capsules may comprise further vitamins selected from vitamin A, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B9 (folic acid or folate), vitamin C (ascorbic acid), vitamin D (calciferols), vitamin E (tocopherols and tocotrienols) and vitamin K (quinones) or minerals selected from sulfur, iron, chlorine, calcium, chromium, cobalt, copper, magnesium, manganese, molybdenum, iodine, and selenium.

[0141] Example 13: Capsules comprising a Bacillus subtilis and dipeptides as a prebiotic and an enteric coating

[0142] The capsules as prepared in example 12 were coated with an enteric coating composition.

TABLE-US-00003 TABLE 3 Coating composition Content Dry based on Weight Content based substance coating gain on capsule Compound [g] [%] [%] [%] EUDRAGUARD ® 40.8 36.9 8.2 6.7 biotic HPMC 43.1 39.0 8.6 7.1 Talc 20.4 18.4 4.0 3.3 Polyethylene 4.3 3.9 0.9 0.7 glycol Triethyl citrate 2.0 1.8 0.4 0.3

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