PREBIOTIC COMPOSITIONS

Abstract

The invention discloses preparations comprising L-rhamnose, and the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the form of either free fatty acids, fatty acid salts having an organic counter ion selected from lysine, arginine, ornithine, choline and mixtures of the same, or mixtures of free fatty acids and omega-3 fatty acid salts having an organic counter ion selected from lysine, arginine, ornithine, choline and use of a preparation as a feed or food supplement or in pharmaceutical compositions.

Claims

1. A preparation, comprising L-rhamnose, and omega-3 fatty acids, wherein the omega-3 fatty acids are eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the form of free fatty acids, fatty acid salts having at least one organic counter ion selected from the group consisting of lysine, arginine, ornithine and choline, or a mixture of free fatty acids and fatty acid salts having an organic counter ion selected from the group consisting of lysine, arginine, ornithine and choline.

2. The preparation of claim 1, wherein L-rhamnose is either in its monomeric form or contained in a naturally occurring or synthetic polymer.

3. The preparation of claim 2, wherein L-rhamnose is a contained in a naturally occurring or synthetic polymer, and the naturally occurring or synthetic polymer is pectin.

4. The preparation of claim 1, wherein EPA and DHA are in the form of fatty acid salts having at least one organic counter ion selected from the group consisting of lysine, arginine, ornithine and choline.

5. The preparation of claim 1, further comprising at least one selected from the group consisting of a probiotic strain a prebiotic, an amino acid and an amino acid salt.

6. The preparation of claim 1, further comprising at least one selected from the group consisting of pectin, rhamnogalacturonan, laminarin, galactomannan, barley β-glucan, pyrodextrin, pullulan, arabinoxylan, inulin, fructo-oligosaccharides, and galacto-oligosaccharides.

7. The preparation of claim 1, comprising at least 10 weight-% of omega-3 fatty acid.

8. The preparation of claim 1, comprising at least 10 weight-% of L-rhamnose.

9. A pharmaceutical or nutraceutical dosage form, comprising the preparation of claim 1, wherein the dosage form is a tablet or capsule and a total weight of L-rhamnose, EPA and DHA is not more than 5 g.

10. A pharmaceutical or nutraceutical dosage form, comprising the preparation claim 1, wherein the dosage form comprises granules, sprinkles or sachets and a total weight of L-rhamnose, EPA and DHA is not more than 50 g.

11. The pharmaceutical or nutraceutical dosage form of claim 9, further comprising a colon-specific delivery system.

12. The pharmaceutical or nutraceutical dosage form of claim 11, wherein the colon-specific delivery system comprises a coating, and the coating comprises pectin or a pectin salt.

13. A feed- or foodstuff composition, comprising the preparation of claim 1 and an additional feed or food ingredient.

14. A feed or food supplement or a pharmaceutical composition, comprising: the preparation of claim 1.

15. A medicament, comprising the preparation of claim 1.

16. A A method for treating a disease or disorder selected from the group consisting of hyperlipidemia, hypercholesterolemia, non-alcoholic fatty liver, hepatitis, type 2 diabetes, prediabetes, glucose intolerance, arteriosclerosis, other vascular diseases, obesity, adipositas, and multiple sclerosis, the method comprising: administrating the preparation of claim 1 to a subject in need thereof.

Description

WORKING EXAMPLES

[0039] Intestinal Screening Model

[0040] To determine the effect of a EPA/DHA lysine salt (EPA/DHA-Lys) 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 six 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 [18]. These standard adult gut microbiota sets were stored at −80° C. in 12% glycerol.

[0041] The intestinal microbiota was cultured in vitro in modified standard ileal efflux medium (SIEM), the composition of which was described by Minekus et al. [19]. All components were supplied by Trititium Microbiology (Veldhoven, The Netherlands). The pH of the medium was adjusted to 5.8.

[0042] For the I-screen fermentations, the pre-cultured standardized fecal inoculum was diluted 50 times in modified SIEM. EPA/DHA-Lys was introduced into the I-screen to final concentrations of 1.4 mg/ml and 1.5 mg/ml, respectively; omega-3 ethyl ester and fish oil at 1.4 mg/ml each. Inulin was added as a control at a final concentration of 4 mg/ml. 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. All experiments were carried out in triplicates.

[0043] SCFA Analysis

[0044] For the analysis of short-chain fatty acids in exposed material from the I-screen, samples were centrifuged (˜4000 g, 5 min), clear supernatant was filter sterilized (0.45 μm) and a mixture of formic acid (20%), methanol and 2-ethyl butyric acid (internal standard, 2 mg/ml in methanol) was added. A 3-μL sample with a split ratio of 75.0 was injected on a GC-column (ZB-5HT inferno, ID 0.52 mm, film thickness 0.10 μm; Zebron; Phenomenex, USA) in a Shimadzu GC-2014 gas chromatograph. SCFA parameters analyzed were: acetic acid and propionic acid.

[0045] Polyunsaturated Fatty Acid Compositions

[0046] In the examples for the present invention, different polyunsaturated fatty acid compositions were used. Different omega-3 fatty acid salts having an organic counter ion selected from the basic amino acids lysine, arginine and ornithine were prepared. The omega-3 fatty acids Eicosapentaenoic acid (C20:5w3c) (EPA) and Docosahexaenoic acid (C22:6w3c) (DHA) are present in a ratio of around 2:1 (ratio EPA:DHA).

[0047] The omega-3 lysine salt (omega-3-lys) contains around 32 weight-% of L-lysine and around 65 weight-% of polyunsaturated fatty acids. The major polyunsaturated fatty acids in the composition are the omega-3 fatty acids Eicosapentaenoic acid (C20:5w3c) (EPA) and Docosahexaenoic acid (C22:6w3c) (DHA), summing up to around 58 weight-% of the composition. The composition also contains minor amounts of Docosaenoic acid isomer (incl. erucic acid) (C22:1), Docosapentaenoic acid (C22:5w3c) and of the omega-6 fatty acids Arachidonic acid (C20:4w6) and Docosatetraenoic acid (C22:4w6c).

[0048] The omega-3 arginine salt (omega-3-arg) contains around 35 weight-% of L-arginine and around 64 weight-% of polyunsaturated fatty acids. The major polyunsaturated fatty acids in the composition are the omega-3 fatty acids Eicosapentaenoic acid (C20:5w3c) (EPA) and Docosahexaenoic acid (C22:6w3c) (DHA), summing up to around 49 weight-% of the composition. The composition also contains minor amounts of Docosaenoic acid isomer (incl. erucic acid) (C22:1), Docosapentaenoic acid (C22:5w3c) and of the omega-6 fatty acids Arachidonic acid (C20:4w6) and Docosatetraenoic acid (C22:4w6c).

[0049] The omega-3 ornithine salt (omega-3-orn) contains around 29 weight-% of L-ornithine and around 70 weight-% of polyunsaturated fatty acids. The major polyunsaturated fatty acids in the composition are the omega-3 fatty acids Eicosapentaenoic acid (C20:5w3c) (EPA) and Docosahexaenoic acid (C22:6w3c) (DHA), summing up to around 54 weight-% of the composition. The composition also contains minor amounts of Docosaenoic acid isomer (incl. erucic acid) (C22:1), Docosapentaenoic acid (C22:5w3c) and of the omega-6 fatty acids Arachidonic acid (C20:4w6) and Docosatetraenoic acid (C22:4w6c).

Example 1: EPA/DHA-Lys Modulates the Production of Acetate and Propionate by Human Fecal Microbiota

[0050] The effect of different omega-3 fatty acid forms on production of the SCFAs acetate, propionate and n-butyrate by human fecal microbiota was analyzed, which is shown in table 1.

TABLE-US-00001 TABLE 1 Omega-3-lys has unique effects on SCFA production by microbiota. Effects of Omega-3-lys, Omega-3 fish oil, Omega-3 ethyl ester, and inulin on concentrations of acetate, propionate, and n-butyrate after 24 h incubation in human fecal microbiota are given as change in mM compared to a control sample (controls had ~40 mM acetate and ~8 mM propionate after the 24 h cultivation period). Values are given as means of triplicate experiments. Compound Acetate Propionate n-Butyrate Omega-3-lys −3.21 +1.38 −0.86 Omega-3 ethyl ester −2.92 −0.02 −1.26 Fish oil −2.30 −0.17 −0.60 Inulin +5.87 +0.03 +0.46

[0051] The lysine salt of omega-3 (omega-3-lys), but not the commonly used esterified omega-3 derivatives omega-3 ethyl ester (EE) or fish oil, as well as the prebiotic control substance inulin, increased the production of propionate by intestinal microbiota. In parallel, omega-3-lys caused a stronger decrease of acetate production than omega-3 EE or fish oil.

Example 2: Omega-3-Lys, -Arg, and -Orn Salts Increase Propionate Formation and Propionate-to-Acetate Ratio in a Human Intestinal Microbiota

[0052] The effect of different omega-3 fatty acid salts on production of the SCFAs acetate, propionate and n-butyrate by human intestinal microbiota was analyzed, which is shown in table 2.

TABLE-US-00002 TABLE 2 Effects of omega-3 amino acid salts and controls on even- chain SCFA levels in a human intestinal microbiota. Compounds were applied at the following concentrations: Omega-3 amino acid salts = 1.4 mg/ml; EPA/DHA FFA = 0.96 mg/ml; L-lysine = 0.49 mg/ml; L-arginine = 0.52 mg/ml; L-ornithine = 0.5 mg/ml. Values are given as change in mM compared to a control sample as mean of triplicate experiments. Propionate/acetate Compound Acetate Propionate n-Butyrate ratio Omega-3-lys −1.26 +2.28 +0.06 0.69 Omega-3-arg −0.32 +2.22 +0.43 0.64 Omega-3-orn +4.76 +1.55 +0.27 0.45 FFA −6.19 +0.15 −0.69 0.83 Lys +8.81 +2.12 +0.29 0.40 Arg +8.29 +2.02 +0.71 0.40 Orn +10.15 +1.74 +0.19 0.36

[0053] The effects of different omega-3 amino acid salts omega-3-lysine (omega-3-lys), omega-3-arginine (omega-3-arg) and omega-3-ornithine (omega-3-orn) and their respective controls (free fatty acids (FFA), amino acids lysine (lys), arginine (arg) and ornithine (orn)) on SCFA production by an intestinal microbiota were analyzed. All tested omega-3 salts induced propionate production and increased propionate-to-acetate ratios, with omega-3-lys salt showing strongest effects. The amino acids alone also induced propionate, but at the same time strongly induced acetate, resulting in lower propionate-to-acetate ratios as compared to the omega-3 amino acid salts. Omega-3 FFA alone caused a slight increase of propionate levels and a severe reduction of acetate levels, which resulted in a high propionate-to-acetate ratio. In conclusion, a concomitant increase of propionate production and propionate-to-acetate ratio was only achieved after supplementation with omega-3 amino acid salts, with the lysine salt showing the strongest effect.

Example 3: A Combination of L-Rhamnose and EPA/DHA-Lys Results in Enhanced Production of Propionate, Increase in the Propionate-to-Acetate Ratio, which Further Enhances Over Time, by Human Fecal Microbiota 1

[0054] To confirm the results for omega-3 amino acid salts, using omega-3 lysine as an example, an additional, independent and more advanced model of fermentation by the human colonic microbiota was applied, the TIM-2 model. Next to omega-3 lysine the prebiotic carbohydrate L-rhamnose was also applied.

[0055] TIM-2 Model

[0056] TIM-2 is an abbreviation of TNO's in vitro model-2, an advanced dynamic, computer-controlled in vitro model of the adult human colon. TIM-2 simulates to a high degree the successive dynamic processes in the colon and is predictive for what happens in human individuals [19]. The following in vivo conditions are simulated in this model: body temperature, pH in the lumen, composition and rate of secretion, delivery of a pre-digested substrate from the ‘ileum’ (SIEM), mixing and transport of the intestinal contents, absorption of water and microbial metabolites and presence of a complex, high density, metabolically active, anaerobic fecal microbiota of human origin.

[0057] The model was set-up and inoculated with the microbiota on the first day. Following an overnight adaptation period in SIEM, the test-products were fed at final concentrations of 1.4 mg/ml EPA/DHA-Lys or 1 mg/ml L-rhamnose in SIEM during an intervention period of 72 hrs. Cultivation in SIEM without any additional substance served as control condition.

[0058] Samples were taken from the lumen and the dialysate every 24 hrs (t0, t24, t48 and t72) to study the metabolic activity of the gut microbiota (SCFA production) and changes in composition of the gut microbiota (16S rRNA gene analysis).

[0059] SCFA Analysis

[0060] Samples were analyzed for SCFA by ion-chromatography.

[0061] Determination of Changes in the Gut Microbiota

[0062] Samples for microbiota composition were analyzed by Baseclear (Leiden, the Netherlands), with subsequent bioinformatics analysis by UM using our standard pipeline using the QIIME-package. Briefly, the isolation of genomic DNA from the fecal samples (3 mL lumen) was performed using standard molecular biology kits from ZYMO Research provided by BaseClear (Leiden, The Netherlands). The PCR amplification of the 16S rRNA gene (V3 and V4 regions), the Barcoding and the library preparation were carried out by BaseClear. The sequencing was carried out using the Illumina MiSeq system and later the sequences were converted into FASTQ files using BCL2FASTQ pipeline version 1.8.3. The quality cut was applied based on the quality level of Phred (Phred quality score). QIIME software package (1.9.0) was used for microbial analyses. The sequences were classified using Greengenes (version 13.8) as a reference 16S rRNA gene database. Correlations between Operational Taxonomic Units (OTUs) and metabolites or test-product was investigated using by Spearman correlation for metabolites and Kruskal-Wallis correlations for test-products, respectively, by programming in R, using RStudio.

[0063] FIG. 1 shows the cumulative production of SCFA (given in mmol±SD) of duplicate experiments, based on samples taken from lumen and dialysate at 0, 24, 48, and 72 h. FIG. 1 shows accelerated production of propionate by the test compounds, both individually and in combination, whereby the combination of the two leads to highest levels of propionate as well as relatively lower levels of acetate. Table 3 lists absolute values for each data point shown in FIG. 1 and states the propionate-to-acetate (Pr/Ac) ratios for each treatment after 24, 48, and 72 hours. The order of Pr/Ac for the 72 h treatments is control (SIEM)<L-rhamnose<EPA/DHA-Lys (ω-3)<L-rhamnose+EPA/DHA-Lys (ω-3). Importantly, the combination of L-rhamnose and EPA/DHA-Lys resulted in highest propionate levels as well as Pr/Ac of all test conditions (see tables 3 and 4).

TABLE-US-00003 TABLE 3 Cumulative production of SCFA in mmol ± SD of duplicate experiments, based on samples taken every 24 h from lumen and dialysate. Control ω3 time acetate propionate butyrate Pr/Ac time acetate propionate butyrate Pr/Ac  0 0.00 0.00 0.00  0 0.00 0.00 0.00 24 37.90  11.15  24.37  0.29 24 24.09  17.58  22.88  0.73 48 66.14  23.85  45.34  0.36 48 42.68  35.56  41.23  0.83 72 95.52  36.90  60.99  0.39 72 62.36  52.61  60.29  0.84 SD acetate propionate butyrate time acetate propionate butyrate  0 0.00 0.00 0.00  0 0.00 0.00 0.00 24 0.02 0.18 2.33 24 0.61 1   0.62 48 0.34 1.25 0.86 48 2.76 0.17 0.97 72 0.42 0.16 0.37 72 4.27 0.34 0.69 rhamnose ω3 + rhamnose time acetate propionate butyrate Pr/Ac time acetate propionate butyrate Pr/Ac  0 0.00 0.00 0.00  0 0.00 0.00 0.00 24 25.30  14.30  15.69  0.57 24 23.97  15.58  16.66  0.65 48 56.28  31.64  35.02  0.56 48 50.12  40.68  35.92  0.81 72 81.62  45.41  52.10  0.56 72 76.91  72.80  55.46  0.95 SD acetate propionate butyrate time acetate propionate butyrate  0 0.00 0.00 0.00  0 0.00 0.00 0.00 24 1.53 3.26 0.61 24 0.73 0.28 0.93 48 0.28 4.34 0.30 48 2.16 1.74 0.22 72 0.62 2.78 1.30 72 1.47 2.02 1.15

TABLE-US-00004 TABLE 4 Ratios of SCFA (average and SD) were calculated by summing acetate + propionate + butyrate and expressing the individual SCFA as a percentage of the sum. ratios acetate propionate butyrate Control average 58.3% 9.3% 32.4% SD 0.0% 0.1% 0.1% ω-3 average 58.2% 13.4% 28.4% SD 0.7% 2.1% 1.7% rhamnose average 51.1% 20.8% 28.1% SD 0.6% 1.5% 0.9% ω-3 + rhamnose average 52.0% 24.1% 23.9% SD 0.5% 0.5% 0.0%

Example 5: Time-Dependent Modulation of the Human Fecal Microbiota 1 by L-Rhamnose and EPA/DHA-Lys Individually and in Combination: Expansion of Propionate-Producing Taxa

[0064] The microbiota composition was determined as described under Example 4.

[0065] Synergistic effects of the test compounds were found for the taxa Ruminococcus and Collinsella, as shown in FIG. 2.

[0066] FIG. 2 shows the prevalence of Collinsella and Ruminococcus after 72 h cultivation of human fecal microbiota. T1=EPA/DHA-Lys; T2=L-rhamnose.

Example 6: A Combination of L-Rhamnose and EPA/DHA-Lys Results in Enhanced Production of Propionate, Increase in the Propionate-to-Acetate Ratio, which Further Enhances Over Time, by Human Fecal Microbiota 2

[0067] A second TIM-2 study was performed, using conditions as described under Example 3 but applying a different human fecal microbiota (termed microbiota 2), to assess whether the observed effects are dependent on the initial microbiota composition.

[0068] The results obtained with microbiota 2 demonstrate that it differs from microbiota 1 in terms of composition and activity, reflected by a different production of SCFA in the control group at all time points studied. For example, in microbiota 2 the background propionate production is higher than in microbiota 1. Despite this, all test conditions (EPA/DHA-Lys±rhamnose) increased the levels of propionate compared to the control group. Compared to microbiota 1, propionate induction and Pr/Ac increase by EPA/DHA-Lys was even stronger and exceeded the effect of L-rhamnose. Moreover, the synergistic effect on both readouts by co-supplementation of the compounds was also confirmed. It can be concluded that omega-3 fatty acids, especially EPA/DHA-Lys and L-rhamnose increase propionate levels and Pr/Ac irrespective of the initial composition and activity of a human fecal microbiota and that they synergistically interact with each other therein (tables 5 and 6).

[0069] FIG. 3 shows the cumulative production of SCFA (given in mmol±SD) of duplicate experiments, based on samples taken from lumen and dialysate at 0, 24, 48, and 72 h. T1=EPA/DHA-Lys (ω-3); T2=L-rhamnose.

TABLE-US-00005 TABLE 5 Ratios of SCFA (average and SD) were calculated by summing acetate + propionate + butyrate and expressing the individual SCFA as a percentage of the sum. Acetate Propionate Butyrate Control average 49.4% 19.1% 31.5% SD 0.3% 0.1% 0.4% ω-3 (T1) average 35.7% 30.1% 34.3% SD 0.6% 0.3% 0.9% rhamnose (T2) average 45.7% 25.3% 29.0% SD 2.8% 0.3% 2.6% T1 + T2 average 37.5% 35.5% 27.0% SD 3.4% 0.9% 4.3%

TABLE-US-00006 TABLE 6 Cumulative production of SCFA in mmol ± SD of duplicate experiments, based on samples taken every 24 h from lumen and dialysate. Control ω3 time acetate propionate butyrate Pr/Ac time acetate propionate butyrate Pr/Ac  0 0.00 0.00 0.00 0.00 0.00 0.00 24 37.90  11.15  24.37  0.29 24.09  17.58  22.88  0.73 48 66.14  23.85  45.34  0.36 42.68  35.56  41.23  0.83 72 95.52  36.90  60.99  0.39 62.36  52.61  60.29  0.84 SD acetate propionate butyrate time acetate propionate butyrate  0 0.00 0.00 0.00  0 0.00 0.00 0.00 24 0.02 0.18 2.33 24 0.61 1.00 0.62 48 0.34 1.25 0.86 48 2.76 0.17 0.97 72 0.42 0.16 0.37 72 4.27 0.34 0.69 rhamnose ω3 + rhamnose time acetate propionate butyrate Pr/Ac time acetate propionate butyrate Pr/Ac  0 0.00 0.00 0.00 0.00 0.00 0.00 24 25.30  14.30  15.69  0.57 23.97  15.58  16.66  0.65 48 56.28  31.64  35.02  0.56 50.12  40.68  35.92  0.81 72 81.62  45.41  52.10  0.56 76.91  72.80  55.45  0.95 SD acetate propionate butyrate time acetate propionate butyrate  0 0.00 0.00 0.00  0 0.00 0.00 0.00 24 1.53 3.26 0.61 24 0.73 0.28 0.93 48 0.28 4.34 0.30 48 2.16 1.74 0.22 72 0.62 2.78 1.30 72 1.47 2.02 1.15

Example 7: Time-Dependent Modulation of the Human Fecal Microbiota 2 by L-Rhamnose and EPA/DHA-Lys Individually and in Combination: Expansion of Propionate-Producing Taxa

[0070] The composition of the gut microbiota was determined as described under Example 3. By using Kruskal-Wallis correlation analysis the genus Prevotella was identified to be significantly affected by omega-3 fatty acids, especially EPA/DHA-Lys and by L-rhamnose, both individually and even stronger in combination (FIG. 4). The abundances of different Operational taxonomic unit (OTUs) was correlated with levels of microbial short-chain fatty acids. As can be seen in table 7, a strongly positive correlation between Prevotella and propionate was identified; positive though weaker correlations occurred also for Prevotellaceae, Lactobacillus, and Clostridium. It can be concluded that these taxa mediate the omega-3-fatty acid- and L-rhamnose-dependent induction of propionate production by gut microbiota.

[0071] FIG. 4 shows the prevalence of Prevotella under the different experimental conditions; Prevotella is positively correlated with omegal-3 fatty acids (q-value=0.00054) and L-rhamnose (q-value=0.0060). T1=EPA/DHA-Lys; T2=L-rhamnose. Time points 24, 48, and 72 h were included in the analysis.

TABLE-US-00007 TABLE 7 Rho-correlations between microbial metabolites and abundances of OTUs. Time points 24, 48, and 72 h were included in the analysis. SCFA acetate propionate butyrate OTU −0.32 Actinomyces Bifidobacterium −0.43 −0.37 Coriobacteriaceae −0.44 −0.43 Adlercreutzia −0.46 −0.5 −0.44 Bacteroidales;Other −0.38 −0.49 −0.42 Bacteroides −0.52 −0.6 Parabacteroides 0.35 Prevotellaceae 0.58 Prevotella −0.58 −0.72 −0.41 Rikenellaceae −0.38 −0.41 Bacteroidales;f −0.44 −0.52 Barnesiellaceae −0.44 Butyricimonas −0.33 Odoribacter −0.48 −0.51 −0.58 Paraprevotella 0.54 0.55 0.61 Lactobacillus 0.41 Lactococcus −0.46 Streptococcus −0.38 Turicibacter −0.42 Clostridiales;Other −0.4 Clostridiales;g −0.47 −0.55 Christensenellaceae −0.33 Clostridiaceae;Other 0.39 0.47 Clostridium −0.41 Clostridiaceae;g −0.36 Lachnospiraceae;Other 0.51 Lachnospiraceae;g.sub.— −0.5 Blautia −0.43 Coprococcus −0.53 Dorea −0.41 −0.41 Lachnobacterium −0.47 Lachnospira −0.41 Roseburia −0.51 Ruminococcus Peptostreptococcaceae −0.5 Ruminococcaceae;Other −0.46 −0.54 Ruminococcaceae;g.sub.— −0.32 Faecalibacterium −0.49 Oscillospira −0.55 Ruminococcus −0.32 Dialister −0.43 Mogibacteriaceae −0.58 Erysipelotrichaceae −0.4 Holdemania −0.44 −0.6 Eubacterium −0.44 Bilophila Morganella −0.35 Haemophilus 0.46 Acinetobacter −0.44 Anaeroplasmataceae −0.56 Mollicutes −0.46 Akkermansia

Example 8: Combinations of Polymer-Bound L-Rhamnose (Pectin) and EPA/DHA-Lys Results in Enhanced Production of Propionate by a Human Fecal Microbiota

[0072] In the following, we assessed whether our observations made with monomeric L-rhamnose can be extended to L-rhamnose contained in naturally occurring polymers with prebiotic functions. One such polymer is pectin, which is found as part of the cell walls of dicotyledonous plants and which contains L-rhamnose in the form of rhamnogalacturonan. Venema et al. reported that fruits have similar L-rhamnose contents ranging from 1.5-3% [20]. Two types of pectin were applied, one sourced from citrus and one from apple, and tested their metabolization by a human colonic microbiota to SCFAs, individually and each in combination with omega-3-lys, over a time course of 72 hours.

[0073] Human Intestinal Microbiota Fermentation Model

[0074] Effects of pectin were analyzed using a sophisticated system with standardized human fecal microbiota in a pH buffered and temperature controlled high-throughput analytical system with subsequent SCFA analysis.

[0075] Human fecal microbiota inoculum was received by five days fed-batch cultivation of a microbiota sample from a single healthy adult donor, with cryoprotected aliquots stored at −80° C. Aliquots were revitalized for 46 hrs under standardized conditions and the resulting inoculum supplied with 7.5 mg/ml pectin (origin: either from apple or citrus) and/or 1.4 mg/ml EPA/DHA-Lys (ω-3) (final concentrations in modified SIEM). Samples were taken from supernatants every 24 hrs (t0, t24, t48 and t72) to study the metabolic activity of the gut microbiota (SCFA production). Cultivation in modified SIEM without any additional substance served as a control.

[0076] SCFA Analysis

[0077] Samples were analyzed for SCFA by gas-chromatography.

TABLE-US-00008 TABLE 8 Cumulative production of SCFA in mmol ± SD of triplicate experiments, based on samples taken every 24 h from supernatant. Control ω3 time acetate propionate butyrate Pr/Ac time acetate propionate butyrate Pr/Ac  0 0.00 0.00 0.00  0 0.00 0.00 0.00 24 18.23  5.47 2.35 0.30 24 20.98  6.84 3.22 0.33 48 28.98  9.45 5.67 0.33 48 26.64  9.72 5.07 0.36 72 35.64  8.05 5.52 0.23 72 36.25  12.15  7.07 0.34 SD acetate propionate butyrate time acetate propionate butyrate  0 0.00 0.00 0.00  0 0.00 0.00 0.00 24 0.25 0.07 0.11 24 2.94 1.02 0.54 48 3.16 0.81 0.11 48 2.83 0.67 0.11 72 0.17 5.12 0.17 72 0.67 0.11 0.05 pectin (apple) ω3 + pectin (apple) time acetate propionate butyrate Pr/Ac time acetate propionate butyrate Pr/Ac  0 0.00 0.00 0.00  0 0.00 0.00 0.00 24 44.30  8.14 4.88 0.18 24 39.80  8.68 3.44 0.22 48 56.01  13.99  6.51 0.25 48 50.01  15.70  5.49 0.31 72 58.51  14.04  8.51 0.24 72 53.79  16.87  7.38 0.31 SD acetate propionate butyrate time acetate propionate butyrate  0 0.00 0.00 0.00  0 0.00 0.00 0.00 24 0.83 0.06 0.00 24 3.76 0.17 0.23 48 2.44 0.45 0.11 48 5.50 1.05 0.14 72 2.52 1.05 1.37 72 2.91 0.58 1.11 pectin (citrus) ω3 + pectin (citrus) time acetate propionate butyrate Pr/Ac time acetate propionate butyrate Pr/Ac  0 0.00 0.00 0.00  0 0.00 0.00 0.00 24 36.08  7.11 8.40 0.20 24 35.97  8.10 7.45 0.23 48 43.57  11.16  8.10 0.26 48 47.18  13.95  8.02 0.30 72 54.40  12.51  10.37  0.23 72 49.35  14.62  8.51 0.30 SD acetate propionate butyrate time acetate propionate butyrate  0 0.00 0.00 0.00  0 0.00 0.00 0.00 24 4.11 0.70 0.72 24 0.49 0.72 0.65 48 12.09  1.76 2.44 48 7.49 1.07 0.70 72 1.45 0.23 0.43 72 4.14 0.34 0.81

[0078] Table 8 shows the cumulative production of SCFA (given in mmol±SD) of triplicate experiments, based on samples taken from supernatant at 0, 24, 48, and 72 h. Table 8 shows increased production of propionate by omega-3-lys (ω-3) compared to control at each tested time point (24, 48, 72 h), and consistently higher propionate-to-acetate ratios as found in the control samples.

TABLE-US-00009 TABLE 9 Ratios of SCFA (average and SD) were calculated by summing acetate + propionate + butyrate and expressing the individual SCFA as a percentage of the sum. time: 72 h % Acetate Propionate Butyrate Control average 72.41 16.37 11.22 SD 0.17 0.03 0.21 ω3 average 65.34 21.90 12.75 SD 0.51 0.41 0.10 pectin (apple) average 72.18 17.32 10.50 SD 0.55 0.34 0.20 ω3 + pectin (apple) average 68.92 21.62 9.45 SD 1.56 0.41 1.15 pectin (citrus) average 70.40 16.19 13.41 SD 0.08 0.34 0.26 ω3 + pectin (citrus) average 68.08 20.18 11.74 SD 0.77 1.03 0.26

[0079] In summary, the pectin-treated samples showed higher propionate levels than control after (24, 48, 72 h). Pr/Ac ratios were lower than or similar to control, though, reflecting higher acetate concentrations found in both pectin-treated samples. Importantly, co-incubations of citrus- as well as apple-pectin with omega-3-lys resulted in propionate levels that surpassed those of the single treatments at each time point. Again, Pr/Ac ratios were in between those of the control and single treatments, reflecting the higher contribution of pectins to acetate production (also displayed in the percentage distribution of SCFA shown in table 9).

[0080] Similar results were obtained by using a different SIEM medium with a minimal carbohydrate content.

[0081] These results broaden our discovery, accordingly L-rhamnose, either when applied as a monomer or when contained in a naturally occurring carbohydrate polymer, as exemplified for pectin, has a synergistic effect on omega-3-dependent production of propionate by a human colonic microbiota.

Example 9: Capsules Comprising EPA-DHA Amino Acid Salts and L-Rhamnose as Food Supplement

[0082] The following components (as shown in table 10) were filled in HPMC capsules (size 0).

TABLE-US-00010 TABLE 10 Preparations for filling into HPMC capsules. Compound Capsule I Capsule II Capsule III Omega-3 amino acid* salt 250 mg 50 mg 800 mg L-rhamnose 200 mg 50 mg 800 mg L-ornithine L-aspartate 200 mg 50 mg 800 mg Pectin 250 mg 50 mg 800 mg Choline 82.5 mg  82.5 mg.sup.  82.5 mg  *Amino acids are selected from L-ornithine, L-lysine and L-arginine.

[0083] The capsules may further contain amino acids selected from L-ornithine, L-aspartate, L-lysine and L-arginine.

[0084] The capsules may further contain further carbohydrate ingredients, selected from arabinoxylans, barley grain fibre, oat grain fibre, rye fibre, wheat bran fibre, inulins, fructooligosaccharides (FOS), galactooligosaccharides (GOS), resistant starch, beta-glucans, glucomannans, galactoglucomannans, guar gum and xylooligosaccharides.

[0085] The capsules may further contain one or more plant extracts, selected from ginger, cinnamon, grapefruit, parsley, turmeric, curcuma, olive fruit, panax ginseng, horseradish, garlic, broccoli, spirulina, pomegranate, cauliflower, kale, cilantro, green tea, onions, and milk thistle.

[0086] The capsules may further contain charcoal, chitosan, glutathione, monacolin K, plant sterols, plant stanols, sulforaphane, collagen, hyalurone.

[0087] The capsules may comprise further vitamins selected from biotin, 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, selenium, and zinc.

Example 10: Capsules Comprising EPA-DHA Amino Acid Salts, L-Rhamnose, Enteric Coating

[0088] The capsules as prepared in example 9 were coated with an enteric coating composition as colon-specific delivery system (as shown in table 11).

TABLE-US-00011 TABLE 11 Coating composition Content Content Dry based on Weight based on substance coating gain 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

Example 11: Formulations Comprising EPA-DHA Amino Acid Salts with Pectin-Based Delivery Systems

[0089] The following components (as listed in table 12) were mixed and either filled in HPMC capsules (size 0) or subjected to microencapsulation.

TABLE-US-00012 TABLE 12 Preparations for filling into HPMC capsules or microencapsulation. Capsule/ Capsule/ Capsule/ Compound Mixture I Mixture II Mixture III Omega-3 amino acid* salt 250 mg 50 mg 800 mg L-ornithine L-aspartate 200 mg 50 mg 800 mg Choline 82.5 mg  82.5 mg.sup.  82.5 mg  *Amino acids are selected from L-ornithine, L-lysine and L-arginine.

[0090] Capsules I-III were coated with compositions containing pectin or calcium pectinate.

[0091] Mixtures I-III were microencapsulated with compositions containing pectin or calcium pectinate.

[0092] The coating compositions for capsules or mixtures may further contain pH dependent polymers or biodegradable polymers, preferably selected from chitosan, gelatin, HPMC methyl acrylate-methacrylic acid copolymers, cellulose acetate phthalate (CAP), cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymers, shellac, cellulose acetate trimellitate, sodium alginate, zein.

[0093] The capsules and mixtures may further contain L-rhamnose and ingredients listed in Example 9.

REFERENCES

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