Probiotic <i>Bifidobacterium breve </i>strain and compositions comprising said strain

Abstract

The present invention relates to Bifidobacterium breve deposited as DSM 32356 and compositions comprising said strain. The composition may further comprise at least one other bacterial strain and/or at least one compound which may be an NSAID such as acetylsalicylic acid (aspirin). In a presently preferred embodiment the invention relates to Bifidobacterium breve deposited as DSM 32356 for use in the support of the defense against intestinal tissue damage such as intestinal mucosal breaks or lesions e.g. in connection with NSAID administration such as in connection with administration of acetylsalicylic acid (aspirin). The invention further provides a method of supporting the defense against intestinal tissue damage, the method comprising administering the Bifidobacterium breve strain deposited as DSM 32356 to a subject in need thereof, e.g. to a subject in need of NSAID treatment.

Claims

1. A method of supporting defense against intestinal tissue damage in a subject in need thereof, comprising orally administering to the subject a therapeutically effective amount of bacteria of Bifidobacterium breve strain DSM 32356 deposited at DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ) under accession number DSM 32356.

2. The method of claim 1, wherein the bacteria of strain DSM 32356 are administered in a composition that further comprises a cryoprotectant.

3. The method of claim 1, wherein the bacteria of strain DSM 32356 are administered in composition having a concentration of Bifidobacterium breve strain DSM 32356 of at least 10.sup.6 CFU/g.

4. The method of claim 1, wherein the bacteria of strain DSM 32356 are administered in a composition that further comprises bacteria of at least one other bacterial strain.

5. The method of claim 1, wherein the bacteria of strain DSM 32356 are administered in a composition that further comprises Bifidobacterium infantis bacteria.

6. The method of claim 5, wherein the bacteria of strain DSM 32356 are administered in composition having a total concentration of Bifidobacterium breve strain DSM 32356 and Bifidobacterium infantis bacteria of at least 10.sup.6 CFU/g.

7. The method of claim 1, wherein the bacteria of strain DSM 32356 are administered in a composition that further comprises one or more compounds selected from vitamins, prebiotics, fiber, fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), and human milk oligosaccharides (HMO).

8. The method of claim 1, wherein the bacteria of strain DSM 32356 are administered in a composition that comprises Bifidobacterium breve strain DSM 32356 as the only probiotic component.

9. The method of claim 1, wherein the Bifidobacterium breve strain DSM 32356 is administered in an amount from 10.sup.8-10.sup.11 CFU/day.

10. The method of claim 1, wherein the subject is undergoing NSAID administration of a non-steroidal anti-inflammatory (NSAID) drug.

11. The method of claim 10, wherein the subject is undergoing acetylsalicylic acid (aspirin) administration.

12. The method of claim 10, wherein the method is effective to reduce intestinal mucosal breaks or lesions as compared to a subject undergoing NSAID administration without administration of Bifidobacterium breve strain DSM 32356.

13. A composition comprising bacteria of Bifidobacterium breve strain DSM 32356 in dried, frozen, or freeze-dried form, further comprising an effective amount of a cryoprotectant.

14. The composition of claim 13, wherein the concentration of Bifidobacterium breve strain DSM 32356 in the composition is at least 10.sup.6 CFU/g.

15. The composition of claim 13, further comprising Bifidobacterium infantis bacteria.

16. The composition of claim 13, further comprising one or more compounds selected from vitamins, prebiotics, fiber, fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), and human milk oligosaccharides (HMO).

17. The composition of claim 13, formulated for oral administration.

18. A feed or food product, dietary supplement, or pharmaceutical composition comprising Bifidobacterium breve strain DSM 32356 at an amount of at least 10.sup.6 CFU/g and an effective amount of a cryoprotectant.

19. A method for producing a feed or food product, dietary supplement, or pharmaceutical composition, comprising adding Bifidobacterium breve strain DSM 32356 to the feed, food product, dietary supplement, or pharmaceutical composition in a concentration an amount of at least 10.sup.6 CFU/g.

Description

LEGEND TO FIGURES

(1) FIG. 1

(2) FIG. 1 shows an overview of the gastrointestinal tract, i.e. the stomach, the small intestine comprising the duodenum, jejunum and ileum, and the large intestine (colon).

(3) FIG. 2

(4) FIG. 2 illustrates the transepithelial electrical resistance (TEER) as percentage of baseline over time, dots are means, error bars are SDs (A) and TEER area-under-the-curve (AUC) from 0-15 hrs, bars are means and error bars are SDs (B). ****p<0.0001.

(5) FIG. 3

(6) In vitro cytokine expression of Bifidobacterium breve DSM 32356-stimulated dendritic cells from 4 donors. Data are expressed as mean±SEM in pg/ml. A. Secretion of IL-10, B. Secretion of IL-12p70 C. IL-10:IL-12p70 ratio. ****p<0.0001.

(7) FIG. 4

(8) FIG. 4 shows the clinical trial design and visit overview. Visit 1 was a screening visit. Subjects then entered a 2-week run-in period before baseline measurements were made at visit 2 at which subjects were randomized to active DSM 32356 or placebo treatment. Intake of trial product began the morning after visit 2. Subjects were also instructed to take 300 mg of acetylsalicylic acid (aspirin) daily from the same morning for the first 6 weeks of the DSM 32356/placebo intervention. Pillcam capsule endoscopy was performed at Visits 2-7.

(9) FIG. 5

(10) FIG. 5 shows the assessment of the primary endpoint, area-under-the-curve for the Capsule Endoscopy Lewis Score. FIG. 5a shows curve dynamics for the two arms and FIG. 5b the area-under-the-curve illustrated as mean value bar charts with a p value of <0.05 (*) (error bars being ±SEM).

(11) FIG. 6

(12) FIG. 6 shows the assessment of the first secondary endpoint, area-under-the-curve for the total number of ulcers as assessed by capsule endoscopy. FIG. 6a shows curve dynamics for the two arms and FIG. 6b the area-under-the-curve illustrated as mean bar charts with a p value of <0.05 (error bars being ±SEM).

(13) FIG. 7

(14) FIG. 7 shows mean serum concentrations of Thromboxane B2 (TXB2) per visit (A), AUC ±SEM (B), mean serum concentrations of Prostaglandin E2 (PGE2) per visit (C) and AUC ±SEM (D).

(15) FIG. 8

(16) IFN-γ levels in plasma of subjects belonging to the placebo or Bifidobacterium breve DSM 32356 group. Data are expressed as median +95% CI in pg/ml.

(17) FIG. 9

(18) IL-10 levels in plasma of subjects belonging to placebo or Bifidobacterium breve DSM 32356 group. Data are expressed as median +95% CI in pg/ml.

(19) FIG. 10

(20) IL-6 levels in plasma of subjects belonging to placebo or Bifidobacterium breve DSM 32356 group. Data are expressed as median +95% CI in pg/ml.

(21) FIG. 11

(22) IL-8 levels in plasma of subjects belonging to placebo or Bifidobacterium breve DSM 32356 group. Data are expressed as median +95% CI in pg/rd.

(23) FIG. 12

(24) TNF-α levels in plasma of subjects belonging to placebo or Bifidobacterium breve DSM32356 group. Data are expressed as median +95% CI in pg/ml.

(25) FIG. 13

(26) C-Reactive Protein (CRP) levels in plasma of subjects belonging to placebo or Bifidobacterium breve DSM 32356 group. Data are expressed as median +95% CI in mg/L.

(27) FIG. 14

(28) FIG. 14 shows Box-plot representing the relative abundance in percent of Bifidobacterium breve in stool in the Bifidobacterium breve DSM 32356 treated and placebo groups. The boxed extends from the first quartile (Q1) to the third quartile (Q3) and the line within the box shows the median value. The lower whisker extends to the smallest value within Q1−1.5× inter-quartile range (IQR) and the upper whisker extends to the largest value within Q3+1.5×IQR. Values outside the whiskers are shown as circles.

(29) FIG. 15

(30) FIG. 15 shows a Box-plot with the Bray-Curtis dissimilarity of stool microbial composition for the Bifidobacterium breve DSM 32356 and placebo groups, comparing Visit 2 with later visits (V3-V7). The boxed extends from the first quartile (Q1) to the third quartile (Q3) and the line within the box shows the median value. The lower whisker extends to the smallest value within Q1−1.5× inter-quartile range (IQR) and the upper whisker extends to the largest value within Q3+1.5×IQR. Values outside the whiskers are shown as circles.

(31) FIG. 16

(32) Aspirin and salicylic acid concentrations for the 2 different dilutions rows, A) dilution row A inoculated with Bifidobacterium breve DSM 32356 and B) dilution row B where pH was adjusted to a final pH of 4.5.

EXAMPLES

Example 1

(33) Analysis of Antibiotic Susceptibility and Cytotoxic Activity

(34) Antibiotic susceptibility of DSM 32356 was determined by measuring the minimum inhibitory concentrations (MICs) of a number of antibiotics according to the ISO 10932 IDF 223 international standard.

(35) The test performed was a broth microdilution method using VetMIC Lact-1 and Lact-2 panels (National Veterinary Institute of Sweden, Uppsala, Sweden) and growth in LSM medium (ISO-sensitest medium (Oxoid) supplemented with 10% MRS (de Man, Rogosa and Sharpe) broth (BD Difco 288110, UK) with 0.05% Cysteine hydrochloride (CyHCl) (Merck 102839, Germany for 48 hours at 37° C. under anaerobic conditions with three biological replicates. The range of antibiotics tested complies with the European Food Safety Authority (EFSA) “Guidance on the characterisation of microorganisms used as feed additives or as production organisms” (EFSA Journal 2018, 16:5206) for the 25 Bifidobacterium group.

(36) DSM 32356 was found to be sensitive to all antibiotics relevant for the Bifidobacterium group according to the EFSA guideline (gentamycin, streptomycin, tetracycline, erythromycin, clindamycin, chloramphenicol, ampicillin, and vancomycin) with MIC values below the EFSA 2018 cut-off values.

(37) The genome of DSM 32356 was analyzed for antibiotic resistance genes by screening against the curated database ResFinder which contains more than 2,200 resistance genes (Zankari, E., Hasman, H., Cosentino, S., Vestergaard, M., Rasmussen, S., Lund, O., et al. (2012) Identification of acquired antimicrobial resistance genes. J. Antimicrob. Chemother. 67: 2640-2644). The database was downloaded and imported into CLC Main Workbench version 8.0.1 on Feb. 8, 2019. The genome was screened for resistance genes against the ResFinder database using megaBLAST settings (Expect threshold 10, word size 28). In agreement with DSM 32356 being phenotypically sensitive to all antibiotics tested, no antibiotic resistance genes were identified in the genome of DSM 32356.

(38) DSM 32356 was tested for cytotoxic activity using a Vero cell assay method based on the EFSA guidance “Guidance on the assessment of the toxigenic potential of Bacillus species used in animal nutrition” (EFSA Journal 2014, 12:3665). DSM 32356 was grown at 37° C. under anaerobic conditions in MRS broth (BD Difco 288110, UK) with 0.05% Cysteine hydrochloride (CyHCl) (Merck 102839, Germany. Culture supernatants were isolated after 24 and 48 hours by centrifugation and analyzed for cytotoxicity at Bioneer A/S, Hoersholm, Denmark using the Vero cell assay. DSM 32356 was found to be non-cytotoxic.

Example 2

(39) Intestinal barrier improvements measured in vitro by bacterial ability to increase the electrical resistance across Caco-2 cell monolayer, measured by transepithelial electrical resistance (TEER)

(40) Culturing of Caco-2 Cells

(41) The human intestinal epithelial Caco-2 cell line (DSMZ ACC 169, Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany) was cultured in MEM (Gibco 11095) supplemented with 20% heat inactivated fetal bovine serum (Gibco 10500), 1×MEM non-essential amino acids (Biowest X0557-100) and 1×Pen-Strep-Amp B (Biological Industries 03-033-1B) at 5% CO.sub.2 at 37° C. Caco-2 cell passages 7-30 were used. When the cells were approximately 50% confluent the medium was removed, and the cells were washed twice in Hanks' balanced salt solution (HBSS, lx, Gibco 14175). The cells were trypsinized by adding 2 mL of trypsin (TrypLE Trypsin, Gibco 12604) and left for 4 min in the CO.sub.2 incubator at 37° C. Approximately 10 mL of medium was added to the trypsinized cells, they were counted and a concentration of 100.000 cells/mL in supplemented MEM was prepared. A volume of 500 μL of cell suspension was used to seed each apical compartment of transwell-Clear, Polyester Membranes (12 well, 0.4 μM, Costar, Cat. No. 3460), where after 1.5 mL of supplemented MEM was added to the basolateral compartment. Cells were cultured on the inserts for 21 days with change of medium twice a week. After 22 days the transwells were moved to the CellZscope (NanoAnalytics, Germany). The medium was changed to antibiotics (Abx) free medium adding 1.65 and 0.76 mL of Abx-free medium in the basolateral and apical compartments, respectively. The CellZscope was placed overnight in a CO.sub.2 incubator (5%) at 37° C. and TEER was measured every hour using automated data collection. This overnight measurement of TEER before the experimental start allowed for determination of baseline TEER in each well and as a quality control of a stable electrical resistance.

(42) Preparation of Bifidobacterium Breve

(43) Two days prior to co-incubation with the Caco-2 cells Bifidobacterium breve DSM 32356 and Bifidobacterium breve DSM 20213 were cultured overnight anaerobically in Man Rogosa Sharp (MRS) broth (BD Difco 288110, UK) with 0.05% Cysteine hydrochloride (CyHCl) (Merck 102839, Germany). The day prior to co-incubation the grown cultures were reinoculated in MRS with 0.05% CyHCl (100 μL bacteria solution in 10 mL MRS with 0.05% CyHCl). A dilution row was generated by transferring 1 mL of mixed inoculated culture with 10 mL MRS with 0.05% CyHCl. This was repeated 5 times. The bifidobacteria were cultured anaerobically overnight at 37° C. On the day of co-incubation bacterial growth was evaluated by measuring OD.sub.600 nm and cultures representing late exponential/early stationary phase were selected. For each strain 2 vials representing late exponential/early stationary phase were pooled and centrifuged at 3500×g for 10 min, in order to collect the bacteria pellet. The supernatants were discarded and 20 mL of 37° C. warm HBSS was added and the bacteria were washed and spun down at 3500×g for 10 min. This washing procedure was repeated once followed by a third washing step using 20 mL pre-heated Abx-free medium. Bacterial cells were harvested by spinning at 3500×g for 10 min and the supernatant was discarded. Bacterial cells were resuspended in 5 mL pre-heated Abx-free medium and OD.sub.600 nm was adjusted to 3.8.

(44) Stimulation of Caco-2 cells with Bifidobacterium Breve

(45) In order to stimulate the Caco-2 cells with bifidobacteria, CellZscope measurements were paused, the CellZscope was removed from the CO.sub.2 incubator and 100 μL of apical medium was removed from each transwell. A 100 μL of bacteria solution (final OD.sub.600 nm of 0.5) or media control (MEM) was added to the relevant wells (each in triplicate). The CellZscope were transferred back to the CO.sub.2 incubator and the TEER measurements were resumed and continued overnight. Changes in TEER during bacterial stimulation were calculated relative to the latest value recorded immediately prior to the stimulation (baseline measurement, set to 100%). Area under the curve was calculated for each well and different bacterial stimulation were compared by one-way ANOVA including Tukey's multiple comparisons test.

(46) Results

(47) The ability to increase TEER in Caco-2 monolayers was tested for the following strains: Bifidobacterium breve DSM 32356 and Bifidobacterium breve DSM 20213 and compared to medium control (FIG. 2). Bifidobacterium breve DSM 32356 was found in vitro to enhance the TEER in the polarized monolayer of Caco-2 epithelial cells. Bifidobacterium 5 breve DSM 32356 significantly increased TEER area-under-the-curve (AUC) to a mean of 380 compared to the control group (medium only) that had a mean of 27, showing an increase in mean TEER AUC of 1311%. Also, Bifidobacterium breve DSM 20213 increased mean TEER AUC but only to mean value of 228. When comparing Bifidobacterium breve DSM 32356 to Bifidobacterium breve DSM 20213, Bifidobacterium 10 breve DSM 32356 had a significantly higher mean TEER value (44% higher).

Example 3

(48) Measurement of IL-10/IL-12 Ratio

(49) Preparation of Bifidobacterium Breve

(50) Bifidobacterium breve DSM 32356 was inoculated and cultured anaerobically with AnaeroGen pads (Thermo Scientific AN0025A, UK) at 37° C. in pH 6.5 MRS (de Man, Rogosa and Sharpe broth, Difco, 288110, UK) with 0.05% Cysteine hydrochloride (CyHCl) (Merck 102839, Germany) overnight. A 10-fold dilution series was prepared from the overnight culture and incubated overnight anaerobically at 37° C. A stationary growth phase culture was selected based on measures of optical density at 600 nm (OD600). The bacterial culture was centrifuged for 2 min at 6000 g, washed twice in Hank's Balanced Salt Solution (HBSS, Gibco 14175), and resuspended in antibiotic-free cell culture media (RPMI 1640, Biological Industries, Kibbutz Beit-Haemek, Israel +10% Glycerol) at OD 0.05.

(51) Monocyte-Derived Dendritic Cell (DC) Generation

(52) Human peripheral blood mononuclear cells (PBMCs) were obtained from buffy coats of four healthy donors. Briefly, a density gradient cell separation was performed by centrifugation using Ficoll-Paque PLUS™ (GE Healthcare, Freiburg, Germany). Monocytes were isolated by positive selection for CD14 using magnetic-activated cell sorting with CD14 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured at a density of 2×10.sup.6 cells/mL in complete Dendritic cell (DC) media (RPMI 1640 supplemented with 10 mM HEPES (Sigma-Aldrich, Schnelldorf, Germany), 50 μM2-ME (Sigma-Aldrich, Schnelldorf, Germany), 2 mM L-glutamine (Life Technologies Ltd, Paisley, UK), 10% heat-inactivated fetal bovine serum (Invitrogen, Paisley, UK), 100 U/mL penicillin (Biological Industries, Kibbutz Beit-Haemek, Israel), and 100 μg/mL streptomycin (Biological Industries, Kibbutz Beit-Haemek, Israel) containing 30 ng/mL human recombinant IL-4 and 20 ng/mL human recombinant GM-CSF (both from Sigma-Aldrich, Saint Louis, Mo., USA) at 37° C., 5% CO.sub.2. Fresh complete DC media containing full doses of IL-4 and GM-CSF was added after three days of culture. At day 6, differentiation to immature DCs was verified by surface marker expression analysis (CD11c>90% expression; CD1a>75% expression).

(53) DC Stimulation

(54) Immature DCs were resuspended in fresh complete DC media containing no antibiotics, seeded in 96-well plates at 1×10.sup.5 cells/well, and allowed to acclimate at 37° C., 5% CO.sub.2, for at least one hour before stimulation. DC were stimulated for 20 hours with 20 μL of B. breve (DSM 32356) bacterial culture in total OD=0.01 or DC medium (unstimulated control) at 37° C., 5% CO.sub.2. After stimulation, DC supernatants were sterile filtered through a 0.2 μm Acro-Prep Advance 96-well filter plate (Pall Corporation, Ann Arbor, Mich., USA) for cytokine quantification.

(55) Cytokine Quantification from DCS

(56) Secreted levels of IL-10 and IL-12p70 were quantified using electrochemiluminescence assays (U-plex panel, Meso Scale Discovery, MSD, Rockville, Md., USA) according to the manufacturer's instructions. The mean of lower detection limit for IL-10 and IL-12p70 was 0.04015 and 0.214 pg/ml respectively.

(57) Statistical Analysis

(58) Statistical analysis was performed with GraphPad Prism 7 software (GraphPad Software, La Jolla, USA). Cytokine mean values were compared using Mann-Whitney test. Data are expressed as mean±SEM in pg/ml, or the ratio IL-12p70:IL-10. When a value was below detection limit of detection or below fitting curve, it was replaced by half-limit of detection.

(59) Results

(60) After 20 hours stimulation with Bifidobacterium breve DSM 32356, the supernatants of monocyte-derived dendritic cells were collected, and their cytokine profile was investigated.

(61) Bifidobacterium breve DSM 32356 was shown to activate immune responses from human monocyte derived DCs through the secretion of the T cell-stimulating cytokine IL-12p70 with a mean value of 9.19 pg/ml (FIG. 3B) and the anti-inflammatory IL-10 with a mean value of 313.9 pg/ml (FIG. 3A). Secretion of IL-10 from unstimulated DCs showed a mean value of 0.17 pg/ml, and IL-12p70 was out of detection limit. The IL-10:IL-12p70 ratio of unstimulated dendritic cells was 0.8:1, while it was increased to 166:1 in Bifidobacterium breve DSM 32356 stimulated dendritic cells (FIG. 3C).

Example 4

(62) In Vivo Study of Protection of Bifidobacterium breve DSM 32356 Against Acetylsalicylic Acid Induced Damage of the Small Intestine Mucosa

(63) The present example demonstrates the ability of DSM 32356 to attenuate and/or reverse the effect of NSAID induced small intestinal damage. More specifically, the present trial has demonstrated the ability of DSM 32356 to attenuate and/or reverse low-dose, long-term acetylsalicylic acid (aspirin)-induced deterioration of small intestinal mucosa tissue as assessed by capsule endoscopy in healthy volunteers.

(64) The trial was a single-site, randomised, double-blind, placebo-controlled, two-armed, parallel-group trial in healthy, adult volunteers. The trial investigated the effect of daily intake of the probiotic strain DSM 32356 or placebo when co-administered to daily intake of 300 mg of acetylsalicylic acid (aspirin).

(65) The trial included a run-in period of two weeks duration followed by a six weeks intervention period where DSM 32356/placebo and acetylsalicylic acid (aspirin) was co-administered. After the 6 weeks, DSM 32356/placebo was given for two additional weeks to investigate the potential effects of DSM 32356 on intestinal healing after long-time acetylsalicylic acid (aspirin) use.

(66) Subjects participated in the trial for a total duration of 10 weeks including the run-in phase. Besides the screening visit, the trial consisted of 6 visits during the 8-week intervention period.

(67) After having given their written informed consent, subjects completed the screening procedures to evaluate their eligibility for participation in the trial and completed a run-in period of two weeks duration to washout possible pre-trial probiotics and/or use of medication. After baseline assessments at Visit 2, subjects started daily intake of 300 mg acetylsalicylic acid (aspirin) and were also randomly assigned to 8 weeks daily intake of active Bifidobacterium breve DSM 32356 or placebo product in a ratio of 1:1.

(68) We aimed for 30 subjects to complete the trial in each arm, and assuming a moderate drop-out rate of subjects, we randomized a total of 75 subjects. The trial had in total 66 subjects completing the trial with available efficacy data (ITT population). Table 1 illustrates the subject disposition and Table 2 illustrates and compares the baseline characteristics of the active and placebo arm.

(69) TABLE-US-00001 TABLE 1 Subject disposition during the trial. Data showed in this application is on the Intention to treat population (n = 66) N Subjects screened: 109 Subjects included in run-in phase 75 Subjects randomized 75 Subjects taking trial product 75 Subjects included in intention to treat 66 population

(70) TABLE-US-00002 TABLE 2 Baseline characteristics and accountability of aspirin and trial product of both arms in the intention to treat population. Data is presented as mean ± SD. ITT population DSM 32356 Placebo P-value N 35 31 Age (years) 30.5 ± 6.8  31.2 ± 6.4  0.6675 Gender (m/f) 16/19 14/17 0.9641 Ethnicity (non-Caucasians)  2  0 0.4012 Height (cm) 172.2 ± 12.1  173.4 ± 10.2  0.6662 Weight (kg) 73.5 ± 12.5 72.0 ± 11.4 0.6137 BMI 24.6 ± 2.1  23.8 ± 2.2  0.1235 Blood pressure, Systolic (mm hg) 124.1 ± 7.8  121.6 ± 10.2  0.2756 Blood pressure, Diastolic (mm hg) 78.7 ± 6.9  77.1 ± 7.6  0.3801 alcohol consumption 5.1 ± 3.2 5.5 ± 3.7 0.6919 (“drinks” per week) Accountability of aspirin 98.7 ± 2.4  99.1 ± 1.9  (100% = product subj. should have taken) Accountability of trial product 98.6 ± 2.4  99.0 ± 1.9  (100% = product subj. should have taken)

(71) Criteria for Inclusion was:

(72) Written informed consent

(73) Healthy and without any gastrointestinal discomfort/pain symptoms

(74) Age ≥18-≤40 years of both gender

(75) Sedentary lifestyle (weekly training load below 2 hours within endurance sports)

(76) Willing to abstain from any other probiotic products and/or medication known to alter gastrointestinal function throughout the participation of the trial

(77) Criteria for Exclusion was:

(78) Abdominal surgery which, as judged by the investigator, might affect the GI function (except appendectomy and cholecystectomy)

(79) History of peptic ulcer disease

(80) Any known bleeding disorder

(81) Allergy to aspirin

(82) History of H. pylori disease

(83) Resting diastolic blood pressure 90 mmHg

(84) Resting systolic blood pressure 140 mmHg

(85) A current diagnosis of psychiatric disease

(86) Systemic use of antibiotics, steroids (except contraceptives) or antimicrobial medication in the last 2 months

(87) BMI >27

(88) Daily usage of non-steroidal anti-inflammatory drugs in the last 2 months or incidental use in the last 2 weeks prior to screening (Aspirin, Ibuprofen, Diclofenac, Naproxen, Celecoxib, Mefenamic acid, Etoricoxib, Indometacin)

(89) Usage of medications, except contraceptives, in the last 2 weeks prior to screening

(90) Diagnosed inflammatory gastrointestinal disease and/or irritable bowel syndrome

(91) Lactose intolerance

(92) Any other disease that, by the Investigators discretion, could interfere with the intestinal barrier function of the subject

(93) Participation in other clinical trials in the past 2 months prior to screening

(94) Regular use of probiotics in the last 2 months

(95) Smoking and/or frequent use of other nicotine products

(96) Desire and/or plans on changing current diet and/or exercise regime during the participation of this trial

(97) Use of laxatives, anti-diarrheals, anti-cholinergics and PPI within last 2 months prior to screening

(98) Use of immunosuppressant drugs within last 4 weeks prior to screening

(99) For Women: Pregnancy or lactation.

(100) The Primary endpoint was the effect of 8 weeks oral supplementation of DSM 32356 versus placebo on small intestinal mucosa damage when co-administered to a 6-week acetylsalicylic acid (aspirin challenge) measured as the area-under-the-curve of the capsule endoscopy Lewis score between Visit 2 (randomization) and Visit 7 (end of treatment).

(101) The first of the ranked secondary endpoint was the effect of 8 weeks oral supplementation of DSM 32356 versus placebo on small intestinal mucosa damage when co-administered to a 6-week acetylsalicylic acid (aspirin) challenge measured as the area-under-the-curve for Visit 2-Visit 7 ulcer number from capsule endoscopy.

(102) During the entire trial subjects were instructed to maintain their habitual life style with regards to diet, physical activity level and sleep habits.

(103) Small intestine mucosa deterioration was evaluated using capsule endoscopy as well as indirect with biomarkers in feces and blood samples drawn at visit 2-7.

(104) Intake of probiotic products as well as food and food supplements containing probiotics were not allowed from the screening visit and until the end of the intervention period.

(105) Subjects were not withdrawn from the trial due to single violations, but violations were recorded as protocol deviations.

(106) Any use of illicit drugs (euphorics or stimulants, such as cannabis, opium) was prohibited during the trial.

(107) Subjects were asked to:

(108) Refrain from consuming and food products that may contain live microbes (i.e. fermented milk products)

(109) Refrain from taking any non-steroidal anti-inflammatory drugs (NSAIDs) during the trial period

(110) Refrain from participating in other clinical trials

(111) Refrain from consuming alcohol for two days prior to Visit 2-7

(112) Refrain from consuming spicy food two days prior to Visit 2-7

(113) Refrain from consuming caffeine two days prior to Visit 2-7

(114) Avoid strenuous exercise two days prior Visit 2-7

(115) Fast overnight from (10:00) before attending the Visit 2-7. Clear liquids such as water, soft drinks, de-caffeinated tea/coffee were allowed up until midnight the day before a visit.

(116) Capsule endoscopy is useful to detect small intestine inflammation and damage in the form of villous oedemas, ulcers and stenosis. The capsule endoscopy method was used during visit 2-7 using the Pillcam capsule from Medtronic.

(117) The subjects were asked to fast from 10:00 the day before the visit in order to empty the intestine before the capsule endoscopy procedure. Subjects met fasting in the morning of the visit. The Pillcam signal receiver belt was connected to the subject's upper body before the Pillcam capsule was swallowed with a glass of water. The Pillcam capsule then travels through the ventricle and small intestine and sends data in the form of pictures to the receiver. Data from the capsule was captured for a total of 8 hours after which the capsule is expected to have passed the small intestine. The subjects were allowed to leave the site during the 8-hour capsule recording but were advised not to be physically active during these 8 hours. After 4 hours, subjects were allowed to have a light meal.

(118) In total 5 well-trained gastroenterologists were involved in reviewing and scoring the video data from the capsules. The damage observed in the small intestine from the capsule were divided in the three categories: Villous oedemas, ulcers and stenosis. These three categories were rated both separately and for the primary endpoint also combined into one score, the Lewis Score (Gralnek et al., 2008) (Gralnek I M, Defranchis R, Seidman E, Leighton J A, Legnani P, Lewis B S. Development of a capsule endoscopy scoring index for small bowel mucosal inflammatory change. Aliment Pharmacol Ther. 2008; 27(2):146-154. doi:10.1111/j.1365-2036.2007.03556.x), which is a well-recognized clinical and scientific score for intestinal damage.

(119) The following rules were applied for assessments of data from the capsules:

(120) Safety:

(121) Within a week from the capsule visit, at least one capsule reviewer confirmed that the capsule had passed the small intestine (by verifying from the last pictures that the capsule is in the colon).

(122) Data Quality:

(123) A randomization system was created, so that in parallel to being randomized to Bifidobacterium breve DSM 32356/placebo treatment, subjects were also randomized to 2 of the 5 reviewers to oversee the capsule data from that subject.

(124) The two reviewers that were appointed by the randomization system reviewed all capsule data from all capsule visits for that subject. The two reviewers overseeing capsule data from a subject were also blinded from each other and not being allowed to communicate to each other in any way regarding the capsule data.

(125) Mean capsule data values for those 2 reviewers were calculated. In cases that the data from the two reviewers differed with 4 or more number of ulcers, a third reviewer (the principal investigator) also reviewed the Pillcam dataset for that visit and a mean value of all 3 reviewers were calculated and used as the final data point for that visit.

(126) The results are provided in the figures showing data from the ITT population.

(127) As the results show, the trial met its primary and first secondary endpoint, showing a clear and statistically significant protective effect of the DSM 32356 strain against aspirin-induced intestinal damage as observed on the Lewis score (FIG. 5), and total number of ulcers (FIG. 6).

Example 5

(128) Quantification of Prostaglandin E2 and Thromboxane B2 Concentrations in Blood Samples

(129) Methods

(130) As exploratory endpoints, post-hoc intervention effects on prostaglandin E2 (PGE2) and thromboxane B2 (TBX2) in serum samples downstream of cyclooxygenase (COX) were studied.

(131) After unblinding, a post-hoc lab analysis was performed on 2 biolipids PGE2 and TXB2 downstream from the COX enzyme in fasting serum samples obtained during visit 2 to visit 7. The extraction protocol and LC-MS/MS analysis were performed by Ambiotis SAS (France) as described previously (Le Faouder et al. 2013) Le Faouder, P. et al. LC-MS/MS method for rapid and concomitant quantification of pro-inflammatory and pro-resolving polyunsaturated fatty acid metabolites. J. Chromatogr. B 932, 123-133 (2013). Ambiotis were kept blinded for intervention during analyses and analyses were performed in single measurements.

(132) Results

(133) Intake of acetylsalicylic acid (aspirin) was associated with robust inhibition of serum PGE2 and TXB2 concentrations (FIG. 7). Bifidobacterium breve DSM 32356 intervention did not alter these well-described acetylsalicylic acid-induced changes in metabolites downstream of COX. This suggests that the protective actions of Bifidobacterium breve DSM 32356 do not interfere with the specific cardiovascular-protective properties of acetylsalicylic acid.

Example 6

(134) Systemic Cytokine Profiling

(135) Methods

(136) Cytokine Quantification from Heparin Plasma

(137) Heparin plasma samples from visit 2 to 7 from 31 subjects from the placebo group, and from the active group (consumers of B. breve, DSM 32356) were collected to analyze their cytokine profiles. Secreted levels of IFN-γ, IL-10, IL-12p70, IL-13, IL-1β, IL-2, IL-4, IL-6, IL-8 and TNF-α were quantified using the V-plex human proinflammatory panel 1 from Meso Scale Discovery (Cat. #K15049D) according to the manufacturer's protocol. C-Reactive Protein (CRP) levels were also quantified using a V-plex single assay (Cat. #K151STD) according to the manufacturer's instructions. Inter-plate variation was assessed using a bridge control sample.

(138) Statistical Analysis

(139) Statistical analysis was performed with GraphPad Prism 7 software (GraphPad Software, La Jolla, USA). Cytokine values were compared using a 2-way ANOVA with Sidak's comparison. Data are expressed as median +95% CI in mg/L for CRP and pg/ml for the rest of the cytokines. When a value was below the limit of detection or below fitting curve, it was replaced by half-lower limit of detection (LOD).

(140) Results

(141) No significant differences were found between the placebo and active groups in any of the measured cytokines or CRP. Therefore, consumption of B. breve (DSM 32356) did not induce a systemic immune-response. All the measurements of IFN-γ, IL-10, IL-6, IL-8, TNF-α and CRP were within detection range, and their inter-plate variation values were below 20% (See table 3 and FIGS. 8 to 13). As for IL-12p70, IL-13, IL-1β, IL-2 and IL-4 more than 31.9% of the measurements were below detection limits, which increased inter-plate variation (See table 3).

(142) Table 3. Mean of lower detection limits for the analyzed cytokines, Inter-plate variation based on CV values of the bridge control sample and % of samples below detection limit. * samples for IL-13 measurements were 26 for the placebo and 28 for the active group.

(143) TABLE-US-00003 TABLE 3 Mean Inter-plate variation % samples below Assay LOD (pg/ml) Bridge control CV detection limit IFN-γ 0.259 13.1  0   IL-10 0.016 12.5  0   IL-12p70 0.036 25.2 31.9 IL-13 0.146 33.9 * 77    IL-1β 0.022 58 68.5 IL-2 0.036 63.2 63.2 IL-4 0.008 47.1 76.4 IL-6 0.031 8.4  0   IL-8 0.119 4.8  0   TNF-α 0.055 10.2  0   CRP 1.301 17.5  0  

Example 7

(144) Bifidobacterium breve Recovery and Microbiota Composition of Stool Samples

(145) As exploratory endpoints, post-hoc intervention effects on Bifidobacterium breve abundancy and microbiota composition in stool samples were studied.

(146) After unblinding, a post-hoc lab and bioinformatic analysis was performed on DNA extracted from all obtained fecal samples using a NucleoSpin 96 Soil kit (Macherey-Nagel) and randomly sheared into 350 bp fragments. Libraries were constructed using NEBNext Ultra Library Prep Kit for Illumina (New England Biolabs) and sequenced to at least 30 million read pairs per sample (2×150 bp paired-end Illumina sequencing). Sequencing reads were filtered to remove human and low-quality reads, mapped to the Clinical Microbiomics Human Gut 22M gene catalog, and summarised as a taxonomic relative abundance table as described previously (Nielsen et al. 2014) Nielsen, H. B. et al. Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes. Nat. Biotechnol. 32, 822-828 (2014). The involved parties were kept blinded for intervention during analyses. Changes in relative abundances of taxa between visit 2 and the integral of later time-points was tested using Wilcoxon rank sum test and corrected for multiple comparison using a Bonferroni correction. Similarly, the Bray-Curtis distance between visit 2 and later time-points were compared between the two arms (t-test).

(147) Results

(148) DNA sequencing of all fecal samples obtained showed a clear increase in abundance after randomisation of Bifidobacterium breve in fecal samples obtained from subjects in the Bifidobacterium breve DSM 32356 arm compared to the placebo arm confirming study product compliance (FIG. 14).

(149) Intervention with Bifidobacterium breve DSM 32356 was not associated with significant changes in abundance of specific microbial taxa nor in the changes of the overall microbiome composition as revealed by Bray-Curtis dissimilarity index (FIG. 15).

Example 8

(150) The overall aim with this experiment was to explore if Bifidobacterium breve DSM 32356 influenced the degradation of aspirin in vitro.

(151) Assumptions

(152) In the clinical trial each subject was dosed 300 mg aspirin (acetylsalicylic acid) per day. It is assumed that the dose was taken with approximately 1 dL of water which would correspond to 0.3 g/0.1 L=3 g/L. The molecular weight of aspirin is 180.157 g/mol. Assumed maximal exposure in the gut would then be (3 g/L)/(180.157 g/mol)=0.01665 mol/L=16.65 mM.

(153) To cover a wide concentration range of acetylsalicylic acid the following concentrations were used: 20, 10, 5, 2.5, 1.25, 0.625, 0.3125 and 0 mM of acetylsalicylic acid.

(154) Preparation of Bifidobacterium breve

(155) Two days prior to incubation with the acetylsalicylic acid Bifidobacterium breve DSM 32356 was cultured overnight anaerobically in Man Rogosa Sharp (MRS) broth (BD Difco 288110, UK) with 0.05% cysteine hydrochloride (CyHCl) (Merck 102839, Germany). The next day the grown culture was reinoculated in fresh MRS with 0.05% CyHCl (100 μL bacteria suspension in 10 mL MRS with 0.05% CyHCl). Six dilution rows were generated by transferring 1 mL of mixed inoculated culture to 10 mL MRS with 0.05% CyHCl. This was repeated 5 times. The bifidobacteria were cultured anaerobically overnight at 37° C. On the day of incubation with acetylsalicyl acid bacterial growth was evaluated by measuring optical density at 600 nm (OD.sub.600 nm) and cultures representing late exponential/early stationary phase were selected. For every 2 dilution rows, the 2 vials representing late exponential/early stationary phase were pooled (adding up to 3 vials in total) and together with a medium control vial the tubes were centrifuged at 3500×g for 5 min, in order to collect the bacteria pellet. The supernatants were discarded and 35 mL of MRS with 0.05% CyHCl was added and the bacteria were washed and spun down at 3500×g for 5 min. The washing procedure was repeated once. Further, 15 mL of MRS with 0.05% was added to each for the 3 vials with bacteria pellets. After resuspending the pellets, the 3×15 mL bacteria suspensions were pooled in 1 tube and spun down at 3500×g for 10 min and the supernatant was discarded. A total of 20 mL MRS with 0.05% CyHCl was added and the bacteria was resuspended. OD.sub.600 nm was adjusted to 12 corresponding to a bacterial density of approximately 4.25×10.sup.9 CFU/mL.

(156) Preparation of Acetylsalicylic Acid Solutions

(157) Acetylsalicylic acid, Sigma, A6810, 400 mg was weighed out and dissolved in 100 mL of MRS with 0.05% CyHCl. pH was adjusted to the pH of the media control (pH=6) using 2M NaOH (Diluted from 27% sodium hydroxide solution, Merck). The aspirin solution was diluted 2-fold 6 times in MRS with 0.05% CyHCl to obtain the following concentrations of aspirin 22.2, 11.1, 5.6, 2.8, 1.39, 0.69 and 0.35 mM. All solutions, including a medium control of MRS with 0.05% CyHCl, were sterile filtered.

(158) Incubation of Bifidobacterium breve DSM 32356 with Aspirin

(159) Nine mL of each acetylsalicylic acid solution (22.2, 11.1, 5.6, 2.8, 1.39, 0.69 and 0.35 mM) and the control condition (0 mM) were added to different sterile tubes in duplicate. To one of the dilution rows 1 mL of DSM 32356 suspension was added to each tube to achieve a final concentration of 20, 10, 5, 2.5, 1.25, 0.625, 0.3125 and 0 mM of acetylsalicylic acid and 4.25×10.sup.8 CFU/mL of bacteria. To the second dilution row 1 mL of MRS with 0.05% CyHCl was added and pH was adjusted to 4.5 using approximately 30 μL of HCl (sodium chloride, VWR Chemicals) per tube. Both dilution rows were incubated anaerobically for 24 hrs at 37° C. After incubation, pH was measured in each vial and the solutions were sterile filtered and frozen at −80° C. until metabolite analysis.

(160) Metabolite Analysis

(161) All samples were diluted 10 times in 10 mM ammonium formate (LC-MS grade from VWR Chemicals) with 0.1% formic acid (LC-MS grade from VWR Chemicals). In order to ensure proper quantification of acetylsalicylic acid and salicylic acid, samples A6 and B6 were additionally diluted 2 times, samples A7 and B7 were additionally diluted 4 times and samples A8 and B8 were additionally diluted 10 times.

(162) For quality control, a mixed pooled sample (QC sample) was created by taking a small aliquot from all samples. This QC sample was analysed with regular intervals throughout the sequence.

(163) The analysis was carried out using a UPLC system (Vanquish, Thermo Fischer Scientific) coupled with a high-resolution quadrupole-orbitrap mass spectrometer (Q Exactive™ HF Hybrid Quadrupole-Orbitrap, Thermo Fischer Scientific). An electrospray ionization interface was used as ionization source. Analysis was performed in negative and positive ionization mode. The QC sample was analysed in MS/MS mode for identification of compounds. The UPLC was performed using a slightly modified version of the protocol described by Doneanu et al. 2011 (Doneanu, C. E., Chen, W. & Masseo, J. R. (2011) Waters Application note, 720004042en).

(164) Data was processed using Compound Discoverer 3.0 (Thermo Fischer Scientific). Compound extraction was performed by extracting all features from the raw data followed by a feature detection by grouping features belonging to the same compound. Isotope patterns together with the accurate mass were used to determine the molecular formula. Identification of compounds were performed by one of 3 levels of annotation:

(165) 1) Confident identification by accurate mass, MS/MS spectra and known retention time identified via standards.

(166) 2) Annotation was based on two pieces of information and was divided into two sublevels: a) based on accurate mass and known retention time as obtained from standards analysed on the same system, b) based on accurate mass and MS/MS spectra from an external library.

(167) 3) Annotations on this level was based on library searches using the accurate mass and elemental composition alone. The library searches were performed with an acceptable mass deviation of +/−3 ppm.

(168) Compounds related to the amount of aspirin added were found by calculating the correlation coefficient between peak areas obtained and the acetylsalicylic acid concentration. The behavior of compounds with correlation coefficients higher than 0.9 were analysed in more detail to investigate if they could be acetylsalicylic acid derivatized metabolites.

(169) Results

(170) All the pH adjusted samples (B1-B8) had a similar pH before and after 24 hours of incubation, whereas the samples inoculated with Bifidobacterium breve DSM 32356 all had a pH of 6 prior to incubation and pH of 4-4.5 after incubation (see Table 4).

(171) TABLE-US-00004 TABLE 4 Final Inoculation pH after 24 acetylsalicylic Sample (~4.25 × pH before hrs of acid conc id 10.sup.8CFU/ml) incubation incubation 0 A1 + 6 4 0.3125 A2 + 6 4 0.625 A3 + 6 4 1.25 A4 + 6 4 2.5 A5 + 6 4 5 A6 + 6 4 10 A7 + 6 4-4.5 20 A8 + 6 4-4.5 0 B1 − 4.5 4.5 0.3125 B2 − 4.5 4.5 0.625 B3 − 4.5 4.5 1.25 B4 − 4.5 4.5 2.5 B5 − 4.5 4.5 5 B6 − 4.5 4.5 10 B7 − 4.5 4.5 20 B8 − 4.5 4.5

(172) In all samples containing acetylsalicylic acid most of the acetylsalicylic acid was converted to salicylic acid after 24 hours of incubation with either Bifidobacterium breve DSM 32356 or after being acid-adjusted. When comparing the inoculated dilution row with the acid-adjusted dilution row no major differences were observed between the concentrations of acetylsalicylic acid and salicylic acid in the two sets of samples (FIG. 16).

(173) Importantly, the concentrations of aspirin and salicylic acid, the two active compounds responsible for the therapeutic effects of aspirin, were not different between the inoculated and the acid adjusted dilution rows. When comparing added acetylsalicylic acid to detected peak areas several compounds seem to correlate and have increased peak areas when more acetylsalicylic acid was present. However, in the inoculated samples the bacteria seem to consume a number of compounds present in the media, an ability which is inhibited when increasing concentrations of aspirin are added due to inhibited bacterial growth. This inhibited growth is probably the underlying cause of the observed correlations, supported by the fact that these compounds are present in the same amount in the samples with high aspirin in the A sample set as in the B sample set. In conclusion, Bifidobacterium breve DSM 32356 does not appear to degrade acetylsalicylic acid.