Probiotic Bifidobacterium breve strain and composition 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 currently preferred embodiment, the present invention relates to Bifidobacterium breve deposited as DSM 32356 for use in supporting defence against intestinal tissue damage, such as rupture or damage of the intestinal mucosa, e.g. associated with NSAID administration, such as associated with acetylsalicylic acid (aspirin) administration. The present invention further relates to a method of supporting defence against intestinal tissue damage, the method comprising administering a strain of Bifidobacterium breve deposited as DSM 32356 to a subject in need thereof, e.g. to a subject in need of NSAID treatment.

Description

Technical Field

[0001] The present invention relates to a strain of Bifidobacterium breve deposited as DSM 32356 and compositions comprising said strain. In a currently preferred embodiment, the present invention relates to the use of a strain of Bifidobacterium breve deposited as DSM 32356 for supporting protection against intestinal tissue damage in a subject in need thereof, for example during NSAID administration. Background Art

[0002] Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used throughout the world, both as prescription drugs and as "over-the-counter" preparations. They are commonly used for the short-term treatment of many common pain-related conditions, including headaches and menstrual pain, and for the long-term treatment of chronic inflammatory diseases, such as osteoarthritis, rheumatoid arthritis, ankylosing spondylitis and gout.

[0003] Long-term use of low dose NSAIDs, usually limited to 75-325 mg per day, is also recommended and prescribed for its cardiovascular protective properties. NSAIDs, such as acetylsalicylic acid (aspirin), when used daily have been shown to be beneficial in reducing the risk of heart attack, clot-related stroke and other blood flow problems in patients who have cardiovascular disease or have already had a heart attack or stroke.

[0004] Furthermore, increasing evidence suggests that NSAIDs may be valuable for the prevention of cancer and neurodegenerative diseases and for the treatment of other inflammatory conditions.

[0005] Due to their wide range of indications and therapeutic properties, NSAIDs are some of the most commonly used pharmaceutical agents in the world. In 2010, approximately 30 million American adults used NSAIDs regularly. It is estimated that more than one-third of the American adult population (including 80% of those with known cardiovascular disease) regularly use low-dose NSAIDs.

[0006] However, NSAIDs, such as low doses of acetylsalicylic acid (aspirin) are used in association with gastrointestinal (GI) damage, and the extent of damage depends on dosage, duration of treatment, concomitant medication, and patient risk profile. Accordingly, acetylsalicylic acid (aspirin) has been used as a major cause of peptic ulcer bleeding in developed countries and is associated with a 2- to 4-fold increased risk of upper GI bleeding, and increases with the use of concomitant medication. Generally speaking, compared to non-users, patients taking NSAIDs experience upper GI bleeding and perforation of up to 4.7 times of relative risk. The strategy for preventing the upper GI complications associated with the use of NSAIDs includes proton pump inhibitors (PIPs) and synthetic prostaglandins, etc.

[0007] Figure 1 An overview of the gastrointestinal tract, namely the stomach; the small intestine consisting of the duodenum, jejunum, and ileum; and the large intestine (colon), is shown.

[0008] Live bacteria formulated as probiotics may provide an alternative to prevent or at least reduce the adverse side effects of NSAIDs such as acetylsalicylic acid (aspirin). Gotteland et al., 2001 (Gotteland M, Cruchet S, Verbeke S. Effect of Lactobacillus ingestion on the gastrointestinal mucosal barrier alterations induced by indomethacin in humans. Aliment Pharmacol Ther. 2001; 15: 11-7) concluded that regular intake of live Lactobacillus rhamnosus GG (LGG) may have a protective effect on the integrity of the gastric mucosal barrier against indomethacin-induced damage, but not at the intestinal level.

[0009] According to Wallace, 2013 (Wallace, JL, Mechanisms, prevention and clinical implications of nonsteroidal anti-inflammatory drug-enteropathy, World J Gastroenterol. 2013, March 28; 19(12): 1861-1876), the prevalence and clinical significance of NSAID enteropathy are still far from being recognized. NSAID-induced enteropathy and bleeding occur more frequently than NSAID-induced gastropathy. Significant small intestinal damage and bleeding can be observed in about 70% of long-term NSAID users and in most patients where the damage is subclinical.

[0010] Unlike the case for NSAID gastropathy, there is no proven preventive therapy for NSAID-induced intestinal disease, and the pathogenesis is poorly understood. It occurs more frequently than the lesions caused by these drugs in the upper gastrointestinal tract, but is far more difficult to diagnose and treat.

[0011] Although the pathogenesis of NSAID enteropathy is still not fully understood, it is clear that bacteria, bile, and the enterohepatic circulation of NSAIDs are all important factors. Inhibitors of gastric acid secretion significantly exacerbate NSAID enteropathy, and this effect is due to significant changes in the intestinal microbiome (Syer, SD, Blackler, RW, Martin, R., de Palma, G., Rossi, L., Verdu, E., Bercik, P., Surette MG, Aucouturier, A., Langella P., Wallace, JL, NSAID enteropathy and bacteria: a complicated relationship, J Gastroenterol (2015), 50: 387-393. doi 10.1007s00535-014-1032-1).

[0012] With improved detection methods, such as video capsule endoscopy, physicians are increasingly aware that small bowel damage caused by NSAIDs is much more common and more severe than previously realized.

[0013] Several studies have demonstrated the beneficial effects of probiotics in experimental models of NSAID bowel disease. There have also been studies that have demonstrated the effects of a single probiotic (Lactobacillus casei) (Endo H, Higurashi T, Hosono K, et al. Efficacy of Lactobacillus casei treatment on small bowel injury in chronic low-dose aspirin users: A pilot randomized controlled study. J Gastroenterol. 2011; 46(7):894-905. doi:10.1007 / s00535-011-0410-1) or a mixture of probiotics (Montalto M., Gallo A, Curigliano V, D'Onofrio F, Santoro L. Covino M., Dalvai S, Gasbarini A. and Gasbarrini G. Clinical trial: the effects of a probiotic mixture on non-steroidal anti-inflammatory drug enteropathy-a randomized, double-blind, cross-over, placebo-controlled study. Aliment Phamacol Ther. 2010;32:209-214) against NSAID-induced small intestinal damage.

[0014] Montalto et al., 2010, tested a cocktail VSL#3 consisting of four strains of Lactobacillus (Lactobacillus casei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus bulgaricus), three strains of Bifidobacterium (Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium infantis), and one strain of Streptococcus salivarius subsp. thermophilus). Treatment with VSL#3 before and during indomethacin therapy was found to significantly reduce fecal calreticulin (FCC) in healthy subjects relative to placebo against NSAID-induced small intestinal damage. Capsule endoscopy was not performed in this study.

[0015] Endo et al., 2011 conducted a clinical trial in which a comprehensive analysis of the small intestine (CE score) was performed using wireless capsule endoscopy in twenty-five patients, including 13 in the Lactobacillus casei group and 12 in the control group. At the 3-month evaluation, a significant reduction in the number of mucosal ruptures and CE scores was observed in the Lactobacillus casei group compared to the results in the control group.

[0016] US 2014 / 072543 titled "Composition comprising probiotic bacteria capable of restoring the barrier effect of the stomach which is lost during pharmacological treatment of gastric hyperacidity" (Mogna Giovanni) claims a pharmaceutical or food composition or supplement comprising at least one bacterial strain belonging to the following species: Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus fermentum, Lactobacillus lactis, Lactobacillus pentosus, Bifidobacterium adolescentis, Bifidobacterium angularis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium pseudocatenulum and Streptococcus thermophilus, which is capable of colonizing the stomach at a pH value comprised between 4.5 and 5 and is capable of producing bacteriocins and / or metabolites and / or hydrogen peroxide, the pharmaceutical food composition or supplement being in a form suitable for administering the at least one bacterial strain to the stomach of a subject.

[0017] No experimental data are provided in this application.

[0018] WO 2013 / 034974 (Mogna et al.) entitled "Composition comprising N-acetylcysteine ​​and / or microencapsulated gastroprotected lysozyme in association with probiotic bacteria capable of restoring the stomach's own barrier effect which is lost during the pharmaceutical treatment of gastric hyperacidity" claims a pharmaceutical composition or dietary composition or supplement or medical device for treating patients taking medication to reduce or treat gastric hyperacidity, comprising or, alternatively, consisting of at least one strain belonging to one or more species selected from the group comprising or alternatively consisting of:

[0019] Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus salivarius, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus fermentum, Lactobacillus lactis, Lactobacillus pentosus, Bifidobacterium adolescentis, Bifidobacterium angularis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium pseudocatenulum and Streptococcus thermophilus associated with N-acetylcysteine ​​and / or lysozyme; the strain is capable of colonizing the stomach at a pH comprised between 4.0 and 5.5 and is capable of producing bacteriocins and / or metabolites and / or oxygenated water.

[0020] The application includes the results of a preliminary study involving a total of 40 subjects. Based on this study, the application concludes that the combined administration of specific strains of Lactobacillus rhamnosus LR06, Lactobacillus pentosus LPS01, Lactobacillus plantarum LP01 and Lactobacillus delbrueckii LDD01, also including an effective amount of N-acetylcysteine, is able to restore the protective barrier against harmful bacteria, especially at the gastric level. Recently, Suzuki et al., 2017 (Suzuki T, Masui A, Nakamura J, et al. Yogurt Containing Lactobacillus gasseri Mitigates Aspirin-Induced Small Bowel Injuries: A Prospective, Randomized, Double-Blind, Placebo-Controlled Trial. Digestion. 2017; 95(1): 49-54. doi: 10.1159 / 000452361) reported a trial in which 64 users of acetylsalicylic acid (aspirin) who had been taking acetylsalicylic acid (aspirin) for more than 1 month were recruited. Patients received yogurt containing Lactobacillus gasseri or placebo twice a day for 6 weeks. Small intestinal damage was assessed by capsule endoscopy before and after consuming yogurt. The effect of Lactobacillus gasseri on patient symptoms was also assessed before and after 6 weeks of treatment using the frequency table of symptoms of gastroesophageal reflux disease (FSSG) and the gastrointestinal symptom rating scale (GSRS) questionnaires. After 6 weeks, the Lactobacillus gasseri group had significantly fewer small intestinal mucosal ruptures and reddened lesions compared to the placebo group. FSSG and GSRS scores were also significantly elevated in the Lactobacillus gasseri group, but not in the placebo group.

[0021] Syer et al., 2015 demonstrated that once daily administration of Bifidobacterium adolescentis for 5 days prior to initiation of twice daily naproxen administration significantly reduced intestinal ulcers in an animal model. However, not all species of Bifidobacterium were found to be effective for this application. For example, Bifidobacterium longum subsp. longum JCM 1217 was ineffective in the same model.

[0022] Therefore, there remains a need to identify probiotics that can be used to support protection against intestinal tissue damage, for example during NSAID administration. Summary of the invention

[0023] The present invention relates to a strain of Bifidobacterium breve deposited as DSM 32356. This strain has been found to be sensitive to all relevant antibiotics in MIC tests. Consistent with this, no antibiotic resistance genes were identified when analyzed as described in Example 1. Further, the strain was tested for cytotoxicity by the Vero cell assay and found to be non-cytotoxic.

[0024] Based on the in vitro results provided in Example 1, the Bifidobacterium breve strain deposited as DSM 32356 is considered safe.

[0025] The results of the transepithelial electrical resistance (TEER) assay are provided in Example 2. The results indicate that the strain increases barrier integrity.

[0026] Aspirin has been considered to cause serious clinically relevant side effects, such as erosion, ulcer and bleeding of the stomach and small intestine.Except for suppressing the cyclooxygenase that causes the mucosal prostaglandin of reduction level, the use of aspirin also destroys the phospholipid layer present in mucus and epithelial cell surface bilayer, and decouples oxidative phosphorylation.The combination of these processes reduces intestinal permeability, triggers inflammatory pathways and eventually leads to apoptosis, mucosal erosion and ulcer (Bjarnasson etc. (2018) Gastroenterology, 154,500-514).

[0027] Acute administration of aspirin induces a significant increase in both gastric and duodenal and small intestinal permeability in healthy subjects. The mechanism behind this is not fully elucidated, however, in vitro experimental studies suggest that aspirin reduces the expression of the tight junction protein zonula occludens-1 (ZO-1), which is considered one of the key markers of cell-cell contacts and barrier integrity (Montalto, M., Maggiano, N., Ricci, R., Curigliano, V., Santoro, L., Di Nicuolo, F., Vecchio, FM, Gasbarrine, A., Gasbarrini, G. (2004) Digestion, 69 (4), 225-228). This is supported by recent data showing how aspirin induces reduced transepithelial electrical resistance (TEER) and increased flux of fluorescein isothiocyanate-conjugated dextran across Caco-2 cell layers (strongly suggesting increased permeability). Furthermore, the authors also found that in addition to increased formation of reactive oxygen species (ROS) and oxidative stress-induced modifications in ZO-1 protein, it also reduced ZO-1 expression (Fukui, A., Naito, Y., Handa, O., Kugai, M., Tsuji, T., Yoriki, H., Qin, Y., Adachi, S., Higashimura, Y., Mizushima, K., Kamada, K., Katada, K., Uchiyama, K., Ishikawa, T., Takagi, T., Yagi, N., Kokura, S. & Yoshikawa, T. (2012) Am J Physiol Gastrointest Liver Physiol 303, G927-G936).

[0028] Caco-2 cell monolayers represent a well-established cell line derived from human colon adenocarcinoma that is commonly used as a model of intestinal permeability.

[0029] Based on the in vitro results provided in Example 2, in which it was shown that the Bifidobacterium breve strain deposited as DSM 32356 increased the electrical resistance across a Caco-2 cell monolayer as measured by transepithelial electrical resistance, TEER, the strain was considered to be potentially useful for supporting defense against intestinal tissue damage in subjects requiring NSAID treatment, such as acetylsalicylic acid (aspirin).

[0030] Administration of acetylsalicylic acid (aspirin) - even at low doses - has been shown to cause mucosal inflammation and damage to the small intestine (Endo, H., Sakai, E., Kato, T., Umezawa, S., Higurashi, T., Ohkubo, H. & Nakajima (2015) J Gastroenterol, 50, 378-386).

[0031] By administering bacteria that will correspondingly activate immune cells to secrete cytokines, it is possible to modulate the inflammatory state of the gastrointestinal tract. For example, Foligne and colleagues (Foligne, B., Nutten, S., Grangette, C., Dennin, V., Goudercourt, D., Poirot, S., Dewulf, J., Brassart, D., Mercenier, A. & Pot, B. (2007) World J Gastroenterol, 13 (2), 236-243) found a significant correlation between bacterial-mediated protection against experimental colitis in vivo and the cytokine profile induced by bacteria in vitro, especially the IL-10 and interleukin 12 (IL-10 / IL-12) ratio. The authors showed how in vivo protection against trinitrobenzene sulfonate (TNBS)-induced colitis in mice was mediated by a bacterial strain with the ability to induce high levels of the "anti"-inflammatory cytokine IL-10 and low levels of the "pro"-inflammatory cytokine IL-12 in vitro. In contrast, strains inducing a low IL-10 / IL-12 ratio failed to alleviate inflammatory colitis.

[0032] Therefore, in vitro modulation of human inflammatory cells is a useful tool to predict the induction of bacteria-induced pro-inflammatory or anti-inflammatory intestinal environments.

[0033] Example 3 shows that the Bifidobacterium breve strain deposited as DSM 32356 increases the IL-10 / IL-12 ratio indicating anti-inflammatory properties. Accordingly, the strain can be used to combat aspirin-induced small intestinal inflammation.

[0034] Example 4 provides the results of a large clinical trial in which healthy volunteers were challenged with a daily intake of 300 mg acetylsalicylic acid (aspirin) for 6 weeks to induce intestinal damage captured and quantified by capsule endoscopy. In addition to acetylsalicylic acid (aspirin) intake, subjects were also randomized to probiotic or placebo treatment. The primary endpoint of the trial clearly showed a significant protective effect against acetylsalicylic acid-induced intestinal damage in the active probiotic arm compared to the placebo arm, as measured by Lewis score (Gralnek IM, Defranchis R, Seidman E, Leighton JA, Legnani P, Lewis BS. 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).

[0035] Example 5 provides results from the serum concentrations of PGE2 and TXB2 in study subjects in the placebo group versus subjects administered DSM 32356, and shows that the strain does not affect aspirin-induced COX inhibition. This suggests that the protective effect of DSM 32356 does not interfere with the specific cardiovascular properties of acetylsalicylic acid (aspirin).

[0036] Systemic cytokine profiles from blood samples collected 1 day after the last dose are provided in Example 6. No significant differences were found between the placebo and active groups, indicating that there were no systemic effects of DSM 32356.

[0037] Example 7 shows that the relative abundance of Bifidobacterium breve increased during intervention with DSM 32356, indicating study product compliance. The fecal biota composition was further investigated and intervention with Bifidobacterium breve DSM 32356 did not appear to significantly alter the fecal microbiota diversity.

[0038] Example 8 provides the results of co-incubation experiments of DSM 32356 with acetylsalicylic acid and shows that DSM 32356 does not appear to degrade acetylsalicylic acid (aspirin).

[0039] In line with this, the present invention relates to Bifidobacterium breve deposited as DSM 32356 for use in supporting protection against intestinal tissue damage in a subject in need thereof. DETAILED DESCRIPTION

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. Although any methods and materials equivalent or similar to those described herein can be used in the practice of the present disclosure, typical methods and materials are described. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or otherwise clearly contradicted by context.

[0041] In the context of describing the present invention (especially in the context of the following claims), the use of the terms "a" and "an" and "said" and similar indicators is interpreted as covering the singular and plural, unless otherwise indicated herein or clearly contradicted by the context. The terms "include", "have", "include" and "contain" are interpreted as open terms (i.e., meaning "including but not limited to"), unless otherwise recorded. Unless otherwise indicated herein, the description of the range of values ​​herein is only intended to be used as a shorthand method for individually referring to each separate value falling within the range, and each separate value is incorporated into the specification as if it is separately set forth herein. All methods described herein can be performed in any appropriate order, unless otherwise indicated herein or clearly contradicted by the context otherwise. Any and all examples provided herein, or the use of exemplary language (e.g., "such as"), are only intended to better illustrate the present invention and do not limit the scope of the present invention, unless otherwise stated. The language in the specification should not be interpreted as indicating that any unrequired element is necessary for the practice of the present invention.

[0042] The present invention provides an isolated strain of Bifidobacterium breve deposited as DSM 32356, which is sensitive to gentamicin, streptomycin, tetracycline, erythromycin, clindamycin, chloramphenicol, ampicillin and vancomycin in a MIC test when tested as described in Example 1; has no antibiotic resistance genes when analyzed as described in Example 1; and is found to be non-cytotoxic when tested for cytotoxicity by the Vero cell assay as described in Example 1.

[0043] The Bifidobacterium breve strain deposited as DSM 32356 is further capable of (i) increasing the electrical resistance across a Caco-2 cell monolayer measured by transepithelial electrical resistance, TEER, by more than 20% after 10 hours of treatment when tested as outlined in Example 2, wherein the Bifidobacterium breve strain deposited as DSM 32356 is compared to the Bifidobacterium breve strain deposited as DSM 20213; (ii) inducing the secretion of more than 200 pg / ml of IL-10 when co-incubated with human monocyte-derived dendritic cells and (iii) inducing an increase in the IL-10 / IL-12 ratio in monocyte-derived dendritic cells stimulated with DSM 32356 compared to non-stimulated monocyte-derived dendritic cells when tested as outlined in Example 3.

[0044] Finally, in clinical studies, in healthy adults taking NSAIDs, such as acetylsalicylic acid, the strain of Bifidobacterium breve deposited as DSM 32356 has been found to protect intestinal tissue against damage, such as mucosal rupture or damage. The results provided in Example 4 suggest that taking 5 to 10 mg / day of 10 Colony forming units (CFU) of Bifidobacterium breve deposited as DSM 32356 can reduce the risk of ulcers in healthy adults taking a daily NSAID, such as acetylsalicylic acid.

[0045] In the context of the present invention, the term "probiotic component" refers to live microorganisms or freeze-dried microbial cultures, dead microorganisms, fragments of microorganisms and extracts or supernatants of microorganisms that beneficially affect the host when applied to humans or animals (FAO / WHO (2001) Health and nutritional properties of probiotics in foods including milk powder with live lactic acid bacteria. Report of the Joint FAO / WHO Expert Consultation on the evaluation of the health and nutritional properties of probiotics in foods including milk powder with live lactic acid bacteria).

[0046] The present invention provides Bifidobacterium breve deposited under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure on August 9, 2016 under the registration number DSM 32356 at the German Collection of Microorganisms, Inhoffenstr. 7B, D-38124 Braunschweig.

[0047] The present invention relates to the use of Bifidobacterium breve deposited as DSM 32356 as a probiotic.

[0048] The present invention further provides a composition comprising the strain.

[0049] As used herein, a bacterial "strain" refers to a bacterium that remains genetically unchanged when grown or propagated.

[0050] In a further aspect, the present invention provides the Bifidobacterium breve strain deposited as DSM 32356 for use in supporting defences against intestinal tissue damage in a subject in need thereof.

[0051] In this context, the term "intestinal tissue damage" includes one or more areas of erythema, erosion, lesion or mucosal disruption or with villous edema, which may proceed to develop severe damage, such as stricture or necrosis.

[0052] In this context the term "mucosal disruption or injury" is used synonymously with the term "ulcer", as this is the definition of an ulcer that has been used in this trial.

[0053] A currently preferred embodiment of the invention relates to Bifidobacterium breve deposited as DSM 32356 for use during NSAID administration, such as during administration of acetylsalicylic acid (aspirin). Obviously, Bifidobacterium breve deposited as DSM 32356 may also be used to restore or reverse intestinal tissue damage following NSAID administration.

[0054] Thus, Bifidobacterium breve deposited as DSM 32356 may be useful during and after NSAID administration in connection with pain-related conditions including headaches and menstrual pain, and in particular during long-term therapy of chronic inflammatory diseases such as osteoarthritis, rheumatoid arthritis, ankylosing spondylitis and gout.

[0055] A currently further preferred embodiment of the invention relates to Bifidobacterium breve deposited as DSM 32356 for use in reducing disruption or damage to the intestinal mucosa.

[0056] The present invention further relates to a method of supporting defences against intestinal tissue damage, the method comprising administering to a subject in need thereof, such as a subject in need of NSAID treatment, a strain of Bifidobacterium breve deposited as DSM 32356. The composition of the present invention may be used to support defences against intestinal tissue damage in a subject at risk of developing intestinal tissue damage, for example due to planned, or ongoing NSAID administration such as low dose administration of, for example, acetylsalicylic acid (aspirin).

[0057] In a currently preferred embodiment, Bifidobacterium breve deposited as DSM 32356 is used in subjects not treated with PPI / other antacid medications, such as subjects taking low dose acetylsalicylic acid (aspirin) as their only medication.

[0058] Bifidobacterium breve deposited as DSM 32356 can be used for its cardiovascular protective properties during and after administration of an NSAID, such as acetylsalicylic acid (aspirin), to reduce the risk of heart attack, clot-related stroke and other blood flow problems in patients who have cardiovascular disease or who have already had a heart attack or stroke.

[0059] The Bifidobacterium breve deposited as DSM 32356 can be used to prevent cancer and neurodegenerative diseases and to treat other inflammatory conditions during and after NSAID administration, such as acetylsalicylic acid (aspirin). The composition can also be used to support defense against intestinal tissue damage in subjects who have an increased risk of intestinal tissue damage due to other causes, such as coffee intake, stress, radiotherapy or the effects of drugs such as selective serotonin reuptake inhibitors, anticoagulants, alendronate, steroids and immune system suppressants such as chemotherapy. Obviously, the Bifidobacterium breve deposited as DSM 32356 can also be used to restore or reverse intestinal tissue damage after such exposure.

[0060] In a currently preferred embodiment of the present invention, the composition of the present invention is used to reduce intestinal tissue damage, that is, by preventing, reducing or treating intestinal mucosal rupture or damage.

[0061] It is contemplated that Bifidobacterium breve deposited as DSM 32356 may also be used for administration to infants.

[0062] In one embodiment, a composition comprising Bifidobacterium breve deposited as DSM 32356 as described herein may include at least one other bacterial strain and / or at least one compound such as a vitamin, prebiotic, fiber or other compound that may have a beneficial health effect such as fructooligosaccharides (FOS), galacto-oligosaccharides (GOS) or human milk oligosaccharides.

[0063] In a presently preferred embodiment, the composition comprising Bifidobacterium breve deposited as DSM 32356 does not include lysozyme or N-acetylcysteine ​​(NAC).

[0064] For example, the composition of the invention may comprise the bacteria of the Bifidobacterium breve strain deposited as DSM 32356 and at least one other bacterial strain.

[0065] The composition of the present invention may comprise a bacterium of the Bifidobacterium breve strain deposited as DSM 32356 and a bacterium of at least one other bacterial strain, wherein the at least one other bacterial strain is selected from the group consisting of: Lactococcus lactis subsp. lactis biovar. diacetylactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis; any strain belonging to the genus Lactobacillus, including but not limited to Lactobacillus acidophilus, Lactobacillus casei subsp. casei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus lactis subsp. lactis, Lactobacillus rhamnosus, Lactobacillus salivarius; any strain belonging to the genus Bifidobacterium, including but not limited to Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium animalis subsp.lactis), Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium magnum, Bifidobacterium pseudocatenulatum pseudocatenulatum; or from the genus Akkermansia, Anaerostipes, Butyricicoccus, Christensenella, Clostridia, Coprococcus, Dorea, Eubacterium, Faecalibacterium, or Roseburia, or from the family Coriobacteriaceae.

[0066] In a preferred embodiment, the composition does not include Lactobacillus plantarum.

[0067] Therefore, the composition may further include at least one strain of bacteria selected from the group consisting of Bifidobacterium animalis subsp. lactis deposited as DSM 15954, Lactobacillus acidophilus deposited as DSM 13241, Lactobacillus rhamnosus deposited as ATCC 53103, Lactobacillus rhamnosus deposited as ATCC 55826, Lactobacillus reuteri deposited as ATCC 55845, Lactobacillus paracasei subsp. paracasei deposited as ATCC 55544, Lactobacillus paracasei deposited as LMG-17806, Streptococcus thermophilus deposited as DSM 15957, Lactobacillus fermentum deposited as NM02 / 31074, Lactobacillus paracasei subsp. paracasei deposited as CCTCC M204012.

[0068] In a currently preferred embodiment, only one, two, three, four or five different strains are present in the composition according to the invention.

[0069] In one aspect of this embodiment, the composition of the invention comprises bacteria of the strain Bifidobacterium breve, such as the strain Bifidobacterium breve deposited as DSM 32356, and bacteria of the strain Bifidobacterium animalis subsp. lactis, such as the strain Bifidobacterium animalis subsp. lactis deposited as DSM 15954. The composition may comprise bacteria of both strains as the sole probiotic component.

[0070] In another aspect of this embodiment, the composition of the invention comprises bacteria of the strain Bifidobacterium breve, such as the strain deposited as DSM 32356, and bacteria of the strain Lactobacillus rhamnosus, such as the strain deposited as ATCC 53103. The composition may comprise bacteria of both strains as the sole probiotic component.

[0071] In a further aspect of this embodiment, the composition of the invention comprises bacteria of the strain Bifidobacterium breve, such as the strain Bifidobacterium breve deposited as DSM 32356, bacteria of the strain Bifidobacterium animalis subsp. lactis, such as the strain Bifidobacterium animalis subsp. lactis deposited as DSM 15954, and bacteria of the strain Lactobacillus rhamnosus, such as the strain Lactobacillus rhamnosus deposited as ATCC 53103. The composition may comprise bacteria of these three strains as the only probiotic component.

[0072] In yet another aspect of this embodiment, the composition of the invention comprises bacteria of the strain Bifidobacterium breve, such as the bacteria of the strain Bifidobacterium breve deposited as DSM 32356, bacteria of the strain Bifidobacterium infantis, and bacteria of the strain Bifidobacterium longum. The composition may comprise bacteria of these three strains as the only probiotic component.

[0073] In a still further aspect of this embodiment, the composition of the invention comprises bacteria of the strain Bifidobacterium breve, bacteria of the strain Bifidobacterium infantis, bacteria of the strain Bifidobacterium longum, and bacteria of the strain Bifidobacterium bifidum deposited as DSM 32356. The composition may comprise bacteria of these four strains as the only probiotic component.

[0074] In a further aspect of this embodiment, the composition of the invention comprises bacteria of the strain Bifidobacterium breve, bacteria of the strain Bifidobacterium infantis, bacteria of the strain Bifidobacterium longum, bacteria of the strain Bifidobacterium bifidum, and bacteria of the strain Bifidobacterium catenuliferus deposited as DSM 32356. The composition may comprise bacteria of these five strains as the only probiotic component.

[0075] In one embodiment of this aspect, the bacterium of the strain Bifidobacterium breve deposited as DSM 32356 is the only probiotic component in the composition.

[0076] The composition of the invention may include the bacteria in any suitable form for administration to a subject. In a preferred embodiment, the composition of the invention may include the bacteria in a dry form obtainable by freeze drying, spray drying or lyophilization.

[0077] If bacteria are freeze-dried, they are usually mixed with a cryoprotectant before they are freeze-dried in order to obtain high viability. The term "cryoprotectant" used in the context of the present invention refers to a substance that can improve survival during freezing and / or drying and improve the storage stability of bacteria. The cryoprotectant used herein preferably comprises a sugar or a sugar alcohol, such as inositol.

[0078] Sugar can be a monosaccharide, a disaccharide, an oligosaccharide or a polysaccharide, or a mixture of at least two sugars. Available monosaccharides include glucose (also known as dextrose), fructose, ribose and galactose and useful disaccharides include sucrose, trehalose, maltose and lactose, etc. The composition can include one or more monosaccharides or disaccharides, such as one, two or three or even more different sugars.

[0079] As an example, the cryoprotectant may include a mixture of a disaccharide, such as sucrose, and a polysaccharide, such as maltodextrin.

[0080] The cryoprotectant may further include peptides, proteins, protein hydrolysates or mixtures thereof. Examples of peptides and proteins to be used are casein, pea, whey, albumin, glutamic acid or gelatin, and any separation or hydrolysate thereof. Other additives may also be present, for example antioxidants such as sodium ascorbate, sodium citrate, trisodium citrate dihydrate and cysteine ​​hydrochloride. Skim milk powder and yeast extract may also be ingredients.

[0081] In one embodiment, the composition of the invention comprises bacteria of the strain Bifidobacterium breve deposited as DSM 32356 in frozen or freeze-dried form and a cryoprotectant. The cryoprotectant may comprise a sugar. In a particularly preferred aspect of this embodiment, the cryoprotectant may comprise a mixture of a disaccharide, such as sucrose, and a polysaccharide, such as maltodextrin.

[0082] The composition of the present invention may include at least one compound, such as an NSAID. Examples of NSAIDs that may be combined with the Bifidobacterium breve strain deposited as DSM 32356 according to the present invention may be one or more of the following NSAIDs: acetylsalicylic acid (aspirin), ibuprofen, ketoprofen, naproxen sodium, diclofenac potassium, diclofenac sodium, etodolac, flurbiprofen, indomethacin, ketorolac, nabumetone, naproxen, oxaprozin, piroxicam, sulindac, celecoxib, mefenamic acid and etoricoxib. The composition may be formulated as a combination of a bacterial strain or strains and a compound, such as an NSAID such as acetylsalicylic acid (aspirin) or as separate compositions.

[0083] Preferably, the composition of the present invention is administered orally. Thus, the composition is generally in a form suitable for oral administration. The composition may be a solid composition or a liquid composition. The composition may be in unit dosage form. For example, the composition may be a capsule, a lozenge, a pill, a tablet, a soft gelatin capsule, a pouch, a stick, a stick powder, or in a more common composition such as an oil drop, an emulsion or a paste, or in any other suitable carrier determined by a person skilled in the art to be an effective carrier for a living organism.

[0084] The composition may be encapsulated using, for example, a suitable polymeric matrix to improve long term stability and storage of the composition. One skilled in the art will recognize that any suitable encapsulating material or matrix may be used, as well as encapsulation methods and techniques known to those skilled in the art.

[0085] The composition may be included in a dietary supplement or pharmaceutical composition or may be part of a feed product or a food product, such as a fermented dairy product, for example yoghurt or infant formula.

[0086] In a further embodiment, the composition of the invention comprises bacteria of the strain Bifidobacterium breve deposited as DSM 32356, in a unit dosage form as described above or in a more general composition or as part of a dietary supplement or feed product or food product, such as a fermented dairy product, e.g. yoghurt or infant formula.

[0087] In one embodiment of the invention, the composition of the invention can be used as a preventive treatment to support defense against intestinal tissue damage in subjects with increased risk of intestinal tissue damage. In another embodiment of the invention, the composition of the invention can be used as a treatment to support defense against intestinal tissue damage in subjects with intestinal tissue damage.

[0088] As used herein, the terms "treating" and "treatment" refer to alleviating, correcting or otherwise hindering, delaying, curing or reversing the progression of a disease or discomfort or at least one symptom of a disease or discomfort, including all applications that reduce the severity of the disease or discomfort. Therefore, treatment does not necessarily imply that the subject is treated until fully recovered from the disease or discomfort. Similarly, the terms "prophylactic", "preventing", "prevention" and the like refer to any and all applications of preventing intestinal tissue damage in a subject that may cause disease or discomfort.

[0089] As used herein, the term "subject" refers to any mammal, including, but not limited to, livestock and other farm animals (such as cows, goats, sheep, horses, pigs, and chickens), performance animals (such as race horses), companion animals (such as cats and dogs), laboratory test animals, and humans. Typically, the subject is a human.

[0090] In one embodiment of the present invention, at least 1 x 10 6 CFU / day. Preferably, the amount is at least 1x10 6 CFU / day, at least 1x10 7 CFU / day, at least 1x10 8 CFU / day, at least 1x10 9 CFU / day, at least 1x10 10 CFU / day, at least 1x10 11 CFU / day, at least 1x10 12 CFU / day.

[0091] In one embodiment of the present invention, the Bifidobacterium breve of the present invention and at least one other strain, if applicable, may be present in an amount of at least 1×10 6 CFU / day. The preferred amount is at least 1x10 6 CFU / day, at least 1x10 7 CFU / day, at least 1x10 8 CFU / day, at least 1x10 9 CFU / day, at least 1x10 10 CFU / day, at least 1x10 11 CFU / day, at least 1x10 12 CFU / day.

[0092] In one embodiment, the composition of the invention comprises bacteria of the strain Bifidobacterium breve deposited as DSM 32356 in dried, frozen or freeze-dried form, and the composition is in the form of 1×10 8 CFU / day up to 1x10 11 CFU / day.

[0093] In a currently preferred embodiment, the composition of the invention comprises bacteria of the strain Bifidobacterium breve deposited as DSM 32356 in dried, frozen or freeze-dried form as the sole probiotic product, and the composition is present in an amount of 1 x 10 8 CFU / day up to 1x10 11 CFU / day.

[0094] Those skilled in the art will recognize that the compositions disclosed herein can be administered at dosage levels and dosing regimens that are desired depending on the circumstances and conditions of the subject. An appropriate dosage regimen can be determined based on the teachings of this application. The dosage regimen can be adjusted to provide optimal support for intestinal tissue damage in a subject. Those skilled in the art will recognize that the precise amount and rate of administration of the Bifidobacterium breve strain will depend on many factors, such as the age, weight, general health, sex, and dietary requirements of the subject. Based on the teachings herein, those skilled in the art will be able to determine an appropriate dosage regimen on a case-by-case basis through routine trial and experimentation.

[0095] In an exemplary embodiment of the invention, the composition of the invention may be administered daily for at least 1 day. Alternatively, the composition may be administered once or more daily for at least 1 day, 2 days, 4 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or longer.

[0096] Accordingly, the present invention provides a composition according to the invention comprising bacteria of the strain Bifidobacterium breve deposited as DSM 32356 in dried, frozen or freeze-dried form, wherein at 1×10 8 CFU / day up to 1x10 11 The composition is administered at a dose of CFU / day for at least 2 weeks.

[0097] In a separate embodiment, the present invention provides a composition of the present invention comprising bacteria of the strain Bifidobacterium breve deposited as DSM 32356 in dried, frozen or freeze-dried form, wherein the 8 CFU / day up to 1x10 11 The composition is orally administered in an amount of CFU / day for at least 8 weeks.

[0098] In a further aspect, the invention provides a combination therapy comprising a composition comprising a Bifidobacterium breve bacterium deposited as the Bifidobacterium breve strain as DSM 32356 and at least one compound as described herein for co-administration to a subject in need thereof. The composition may be formulated as a combination of a bacterial strain or strains and a compound, such as an NSAID, or as separate compositions.

[0099] In a separate embodiment, the Bifidobacterium breve strain is contained in a solid form, such as a powder, tablet or liquid, in a feed product or a food product such as an infant formula, a health food, a food additive, a dietary supplement, a pharmaceutical or over-the-counter preparation.

[0100] In a preferred embodiment, the strain of Bifidobacterium breve is present in a concentration of at least 10 6 CFU / g concentration. Specifically, the concentration may be at least 1x10 6CFU / g, at least 1x10 7 CFU / g, at least 1x10 8 CFU / g, at least 1x10 9 CFU / g, at least 1x10 10 CFU / g, at least 1x10 11 CFU / g or at least 1x10 12 CFU / g.

[0101] In one embodiment, the strain of Bifidobacterium breve and at least one other strain are present in an amount of at least 10 6 CFU / g concentration. Specifically, the concentration may be at least 1x10 6 CFU / g, at least 1x10 7 CFU / g, at least 1x10 8 CFU / g, at least 1x10 9 CFU / g, at least 1x10 10 CFU / g, at least 1x10 11 CFU / g or at least 1x10 12 CFU / g.

[0102] In a further aspect, the present invention provides a method for producing a feed product or a food product, a dietary supplement or a pharmaceutical composition, comprising producing a bacterium of the strain Bifidobacterium breve deposited under the number DSM 32356, and subjecting it to at least 10 6 CFU / g concentration is added to a food product, dietary supplement or pharmaceutical composition.

[0103] Preservation and Expert Solutions

[0104] The applicant requests that samples of microorganisms deposited for the present application as described below be made available only to experts before the patent grant date.

[0105] Bifidobacterium breve was deposited on August 9, 2016 under the deposit number DSM 32356 at DSMZ-German Collection of Microorganisms, Inhoffenstr. 7B, D-38124 Braunschweig (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig). BRIEF DESCRIPTION OF THE DRAWINGS

[0106] Figure 1

[0107] Figure 1 An overview of the gastrointestinal tract, ie, the stomach, the small intestine including the duodenum, jejunum, and ileum, and the large intestine (colon) is shown.

[0108] Figure 2

[0109] Figure 2 Transepithelial electrical resistance (TEER) as a percentage of baseline over time, points are mean, error bars are SD (A) and TEER area under the curve (AUC) from 0-15 hours, bars are mean and error bars are SD (B) are illustrated. ****p<0.0001.

[0110] Figure 3

[0111] In vitro cytokine expression of dendritic cells stimulated with Bifidobacterium breve DSM 32356 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.

[0112] Figure 4

[0113] Figure 4 The clinical trial design and visit overview are shown. Visit 1 is a screening visit. Subjects then enter a 2-week run-in period and then have baseline measurements at Visit 2, at which time subjects are randomized to either active DSM 32356 treatment or placebo treatment.

[0114] Intake of the test product began the morning after Visit 2. Subjects were also instructed to take 300 mg of acetylsalicylic acid (aspirin) daily starting the same morning, continuing the previous 6 weeks of the DSM 32356 / placebo intervention. Pillcam capsule endoscopy was performed at Visits 2-7.

[0115] Figure 5

[0116] Figure 5 shows the assessment of the primary endpoint, the area under the curve, of the Lewis score for capsule endoscopy. Figure 5a The curvilinear dynamics of the two arms are shown and Figure 5b Shown are areas under the curve interpreted as bar graphs of the mean, p-value < 0.05 (*) (error bars are ± SEM).

[0117] Figure 6

[0118] Figure 6 shows the assessment of the area under the curve for the first secondary endpoint, total number of ulcers as assessed by capsule endoscopy. Figure 6a The curvilinear dynamics of the two arms are shown, and Figure 6b Shown are areas under the curve interpreted as bar graphs of the mean, p-values ​​< 0.05 (error bars ± SEM).

[0119] Figure 7

[0120] FIG. 7 shows the mean serum concentration (A) and AUC±SEM (B) of thromboxane B2 (TXB2) at each visit, and the mean serum concentration (C) and AUC±SEM (D) of prostaglandin E2 (PGE2) at each visit.

[0121] Figure 8

[0122] IFN-γ levels in plasma of subjects belonging to the placebo or Bifidobacterium breve DSM 32356 groups. Data are expressed as median + 95% CI in pg / ml.

[0123] Fig. 9

[0124] IL-10 levels in plasma of subjects belonging to the placebo or Bifidobacterium breve DSM 32356 groups. Data are expressed as median + 95% CI in pg / ml.

[0125] Fig.10

[0126] IL-6 levels in plasma of subjects belonging to the placebo or Bifidobacterium breve DSM 32356 groups. Data are expressed as median + 95% CI in pg / ml.

[0127] Fig.11

[0128] IL-8 levels in plasma of subjects belonging to the placebo or Bifidobacterium breve DSM 32356 groups. Data are expressed as median + 95% CI in pg / ml.

[0129] Fig.12

[0130] TNF-α levels in plasma of subjects belonging to the placebo or Bifidobacterium breve DSM 32356 groups. Data are expressed as median + 95% CI in pg / ml.

[0131] Fig.13

[0132] C-reactive protein (CRP) levels in plasma of subjects belonging to the placebo or Bifidobacterium breve DSM 32356 groups. Data are expressed as median + 95% CI in mg / ml.

[0133] Fig.14

[0134] Fig.14A box plot showing the relative abundance of Bifidobacterium breve in feces in the group treated with Bifidobacterium breve DSM 32356 and the placebo group in percent is shown. The box extends from the first quadrant (Q1) to the third quadrant (Q3), and the line in the box shows the median. The lower whisker extends to the minimum value in Q1, -1.5 x interquartile range (IQR), and the upper whisker extends to the maximum value in Q3, +1.5 x IQR. The values ​​outside the whiskers are shown as circles.

[0135] Fig.15

[0136] Fig.15 Shown is a box plot of the Bray-Curtis dissimilarity of the fecal microbial composition for Bifidobacterium breve DSM 32356 and placebo groups comparing inquiry 2 with subsequent inquiries (V3-V7). The box extends from the first quadrant (Q1) to the third quadrant (Q3) and the line in the box shows the median. The lower whisker extends to the minimum value in Q1 -1.5x interquartile range (IQR) and the upper whisker extends to the maximum value in Q3 +1.5x IQR. The values ​​outside the whiskers are shown as circles.

[0137] Fig.16

[0138] For 2 different dilution rows of aspirin and salicylic acid concentrations, A) dilution row A, inoculated with Bifidobacterium breve DSM 32356 and B) dilution row B, where the pH was adjusted to a final pH of 4.5.

[0139] Example

[0140] Example 1

[0141] Analysis of antibiotic susceptibility and cytotoxic activity

[0142] The antibiotic susceptibility of DSM 32356 was determined by measuring the minimum inhibitory concentration (MIC) of a number of antibiotics according to the ISO 10932 | IDF 223 international standard.

[0143] The tests performed were broth microdilution methods with three biological replicates, using VetMIC Lact-1 and Lact-2 plates (National Veterinary Institute of Sweden, Uppsala, Sweden) and growing anaerobicly for 48 h at 37 °C in LSM medium (ISO-Sensitive Test Medium (Oxoid) supplemented with 10% MRS (de Man, Rogosa and Sharpe) liquid medium (BD Difco 288110, UK) with 0.05% cysteine ​​hydrochloride (CyHCl) (Merck 102839, Germany). For the Bifidobacterium group, the range of antibiotic tests complied with the European Food Safety Authority (EFSA) "Guidelines on the characterization of microorganisms used as feed additives or as production organisms" (EFSA Journal 2018, 16:5206).

[0144] According to EFSA guidelines, DSM 32356 was found to be susceptible to all antibiotics relevant to the Bifidobacterium group (gentamicin, streptomycin, tetracycline, erythromycin, clindamycin, chloramphenicol, ampicillin, and vancomycin) with MIC values ​​below the EFSA 2018 cut-off values.

[0145] The genome of DSM 32356 was analyzed for antibiotic resistance genes by screening the ResFinder database containing 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). On February 8, 2019, the database was downloaded and imported into CLC Main Workbench version 8.0.1. Using megaBLAST settings (expected threshold 10, word length 28), the genome was screened for resistance genes against the ResFinder database. Consistent with the phenotype of DSM 32356 being sensitive to all tested antibiotics, no antibiotic resistance genes were identified in the genome of DSM 32356.

[0146] Based on the EFSA guideline "Guidelines for the evaluation of the toxinogenic potential of Bacillus species for animal nutrition" (EFSA Journal 2014, 12: 3665), the cytotoxic activity of DSM 32356 was tested using the Vero cell test method. DSM 32356 was grown under anaerobic conditions at 37°C in MRS liquid medium (BD Difco 288110, UK) with 0.05% cysteine ​​hydrochloride (CyHCl) (Merck 102839, Germany). After 24 hours and 48 hours, the culture supernatant was separated by centrifugation and analyzed for cytotoxicity using the Vero cell test at Bioneer A / S, Hoersholm, Denmark. DSM 32356 was found to be non-cytotoxic.

[0147] Example 2

[0148] Improvement in the intestinal barrier measured in vitro was achieved by the ability of bacteria to increase the electrical resistance across a Caco-2 cell monolayer as measured by transepithelial electrical resistance (TEER).

[0149] Caco-2 cell culture

[0150] The human intestinal epithelial Caco-2 cell line (DSMZ ACC 169, Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und 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% CO2, 37°C. Caco-2 cells were used for passage 7-30. When the cells were approximately 50% confluent, the medium was removed and the cells were rinsed twice in Hanks balanced salt solution (HBSS, 1×, Gibco 14175). By adding 2ml of trypsin (TrypLE trypsin, Gibco 12604), the cells are trypsinized and retained in a CO2 incubator at 37°C for 4 minutes. About 10ml of culture medium is added to the trypsinized cells, they are counted and a concentration of 100.000 cells / mL is prepared in supplemented MEM. 500μL volume of cell suspension is used to inoculate each apical compartment of transwell-Clear polyester membrane (12 holes, 0.4μM, Costar, item number 3460), after which 1.5ml of supplementary MEM is added to the basolateral compartment. Cells are cultured on inserts for 21 days, and the culture medium is changed twice a week. After 22 days, transwells are moved to CellZscope (NanoAnalytics, Germany). The culture medium is changed to a culture medium without antibiotics (Abx), and 1.65ml and 0.76ml of Abx-free culture medium are added to the basolateral and apical compartments, respectively. The CellZscope was placed in a CO2 incubator (5%) at 37°C overnight and TEER was measured every hour using automated data collection. This overnight measurement of TEER before the start of the experiment allowed for the determination of a baseline TEER in each well and served as a quality control for stable resistance.

[0151] Preparation of Bifidobacterium breve

[0152] Two days before co-incubation with Caco-2 cells, Bifidobacterium breve DSM 32356 and Bifidobacterium breve DSM 20213 were cultured anaerobically overnight in Man Rogosa Sharp (MRS) broth (BD Difco 288110, UK) with 0.05% cysteine ​​hydrochloride (CyHCl) (Merck 102839, Germany). One day before co-incubation, the growing culture was re-inoculated in MRS with 0.05% CyHCl (100 μL bacterial solution in 10 mL MRS with 0.05% CyHCl). Dilution rows were generated by transferring 1 ml of the mixed inoculum culture with 10 mL of 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, the OD 600nm Bacterial growth was assessed and cultures representing late exponential / early stationary phase were selected. For each strain, 2 vials representing late exponential / early stationary phase were combined and centrifuged at 3500×g for 10 minutes to collect the bacterial pellet. The supernatant was discarded and 20 ml of 37°C warm HBSS was added, and the bacteria were rinsed and centrifuged at 3500×g for 10 minutes. This rinse procedure was repeated once, followed by a third rinse step using 20 mL of pre-warmed Abx-free medium. Bacterial cells were harvested by centrifugation at 3500×g for 10 minutes, and the supernatant was discarded. The bacterial cells were resuspended in 5 mL of pre-warmed Abx-free medium and the OD was 4.3447. 600nm Adjusted to 3.8.

[0153] Stimulation of Caco-2 cells with Bifidobacterium breve

[0154] To stimulate Caco-2 cells with bifidobacteria, pause the CellZscope measurement, remove the CellZscope from the CO2 incubator, and remove 100 μL of the apical medium from each transwell. Add 100 μL of the bacterial solution (final OD 600nm 0.5) or culture medium control (MEM) was added to the relevant wells (each in triplicate). The CellZscope was transferred back to the CO2 incubator and the TEER measurement was repeated and continued overnight. The change in TEER during bacterial stimulation was calculated relative to the last test value recorded immediately before stimulation (baseline measurement, set to 100%). The area under the curve was calculated for each well and the different bacterial stimulations were compared by one-way ANOVA including Tukey's multiple comparison test.

[0155] result

[0156] 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 a medium control ( Figure 2 ). It was found that Bifidobacterium breve DSM 32356 enhanced TEER in polarized monolayers of Caco-2 epithelial cells in vitro. Bifidobacterium breve DSM 32356 significantly increased the TEER area under the curve (AUC) to a mean value of 380 compared to the control group (medium only) with a mean value of 27, showing an increase in the mean TEER AUC of 1311%. Moreover, Bifidobacterium breve DSM 20213 increased the mean TEER AUC, but only to a mean value of 228. When comparing Bifidobacterium breve DSM 32356 to Bifidobacterium breve DSM 20213, Bifidobacterium breve DSM 32356 had a significantly higher mean TEER value (44% higher).

[0157] Example 3

[0158] Measurement of IL-10 / IL-12 ratio

[0159] Preparation of Bifidobacterium breve

[0160] Bifidobacterium breve DSM 32356 was inoculated and cultured anaerobically overnight at 37°C in MRS (de Man, Rogosa and Sharpe broth, Difco, 288110, UK) with 0.05% cysteine ​​hydrochloride (CyHCl) (Merck 102839, Germany) at pH 6.5 using AnaeroGen pads (Thermo Scientific AN0025A, UK). A 10-fold dilution series was prepared from the overnight culture and incubated anaerobically overnight at 37°C. The dilutions were expressed based on the optical density at 600 nm (OD 600 ) were selected for the measurement of stationary growth phase cultures. Bacterial cultures were centrifuged at 6000 g for 2 minutes, washed twice in Hank's balanced salt solution (HBSS, Gibco 14175) and resuspended at OD 0,05 in cell culture medium without antibiotics (RPMI 1640, Biological Industries, Kibbutz Beit-Haemek, Israel + 10% glycerol).

[0161] Monocyte-derived dendritic cell (DC) generation

[0162] Human peripheral blood mononuclear cells (PBMCs) were obtained from buffy coats of four healthy donors. Briefly, density gradient cell separation was performed by centrifugation using Ficoll-Paque PLUS™ (GE Healthcare, Freiburg, Germany). Mononuclear cells were isolated by positive selection for CD14 using CD14 microbead magnetic activated cell sorting (Miltenyi Biotec, Bergisch Gladbach, Germany) and 2×10 6 The density of cells / mL was 400 μg / mL in complete dendritic cell (DC) medium (RPMI1640, supplemented with 10 mM HEPES (Sigma-Aldrich, Schnelldorf, Germany), 50 μM 2-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). Beit-Haemek, Israel) at 37°C, 5% CO2. After three days of culture, fresh complete DC medium containing sufficient IL-4 and GM-CSF was added. On day 6, differentiation into immature DCs was confirmed by surface marker expression analysis (CD11c>90% expression; CD1a>75% expression).

[0163] DC stimulation

[0164] Immature DCs were resuspended in fresh complete DC medium without antibiotics at a concentration of 1 × 10 5 Cells / well were seeded in 96-well plates and allowed to adapt for at least one hour at 37°C, 5% CO2 before stimulation. DCs were stimulated for 20 hours at 37°C, 5% CO2 with 20 μL of a total OD=0.01 of Bifidobacterium breve (DSM 32356) bacterial culture or DC medium (non-stimulated control). After stimulation, DC supernatants were sterile filtered through 0.2 μm Acro-Prep Advance 96-well filter plates (Pall Corporation, Ann Arbor, MI, USA) for cytokine quantification.

[0165] Quantification of cytokines from DCs

[0166] The secretion levels of IL-10 and IL-12p70 were quantified using an electrochemiluminescence assay (U-plex plates, Meso Scale Discovery, MSD, Rockville, Maryland, USA) according to the manufacturer's instructions. The mean values ​​of the lower detection limits for IL-10 and IL-12p70 were 0,04015 pg / ml and 0,214 pg / ml, respectively.

[0167] Statistical analysis

[0168] Statistical analysis was performed using GraphPad Prism 7 software (GraphPad Softwar, La Jolla, USA). Cytokine means were compared using the Mann-Whitney test. Data are expressed as mean ± SEM in pg / ml, or ratio IL-12p70:IL-10. When values ​​were below the detection limit of the assay or below the fitted curve, they were replaced by half of the detection limit.

[0169] result

[0170] After 20 hours of stimulation with Bifidobacterium breve DSM 32356, supernatants of monocyte-derived dendritic cells were collected and their cytokine profiles were investigated.

[0171] Bifidobacterium breve DSM 32356 was shown to activate immune responses from human monocyte-derived DCs by secretion of the T cell-stimulated cytokine IL-12p70 with a mean value of 9.19 pg / ml (Fig. 3B) and secretion of the anti-inflammatory IL-10 with a mean value of 313.9 pg / ml (Fig. 3A). The secretion of IL-10 from non-stimulated DCs showed a mean value of 0.17 pg / ml, and IL-12p70 was outside the detection limit.

[0172] The IL-10:IL-12p70 ratio of non-stimulated dendritic cells was 0.8:1, whereas it increased to 166:1 in dendritic cells stimulated with B. breve DSM 32356 (Fig. 3C).

[0173] Example 4

[0174] In vivo study on the protective effect of Bifidobacterium breve DSM 32356 against acetylsalicylic acid-induced small intestinal mucosal damage

[0175] This example demonstrates the ability of DSM 32356 to attenuate and / or reverse the effects of NSAID-induced small intestinal damage. More specifically, this trial has demonstrated the ability of DSM 32356 to attenuate and / or reverse the deterioration of small intestinal mucosal tissue induced by low-dose, long-term acetylsalicylic acid (aspirin) in healthy volunteers, as assessed by capsule endoscopy.

[0176] The trial was a single site, randomized, double-blind, placebo-controlled, two-arm, 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 a daily intake of 300 mg of acetylsalicylic acid (aspirin).

[0177] The trial included a two-week run-in period, followed by a six-week intervention period in which DSM 32356 / placebo and acetylsalicylic acid (aspirin) were co-administered. After 6 weeks, DSM 32356 / placebo was given for another two weeks to investigate the potential effects of DSM 32356 on intestinal healing after long-term acetylsalicylic acid (aspirin) use.

[0178] The subjects participated in the trial for a total duration of 10 weeks, including a run-in period. In addition to the screening visit, the trial consisted of 6 visits during the 8-week intervention period.

[0179] After having given their written informed consent, subjects completed a screening procedure to assess their eligibility for the trial and completed a two-week run-in period to exclude possible pre-trial probiotic and / or medication use. After the baseline assessment at Visit 2, subjects started a daily intake of 300 mg acetylsalicylic acid (aspirin) and were also randomized in a 1:1 ratio to 8 weekly intakes of either active Bifidobacterium breve DSM 32356 or a placebo product.

[0180] Our goal is to complete 30 subjects in each arm, and assuming a moderate dropout rate of subjects, we will randomize a total of 75 subjects. A total of 66 subjects completed the trial with available efficacy data (ITT population). Table 1 illustrates the subject placement and Table 2 illustrates and compares the baseline characteristics of the active arm and placebo arm.

[0181] Table 1

[0182] Subject placement during the trial. The data presented in this application relate to the willing-to-treat population (n=66)

[0183] N Subject Screening: 109 Subjects included in the run-in period 75 Randomized subjects 75 Subjects taking the test product 75 Subjects included in the intended treatment population 66

[0184] Table 2

[0185] Baseline characteristics and management responsibilities for aspirin and trial products in the two arms in the intended-to-treat population. Data are presented as mean ± SD.

[0186]

[0187] The inclusion criteria were:

[0188] Written informed consent

[0189] Healthy and without any gastrointestinal discomfort / pain symptoms

[0190] Age ≥18-≤40 years old, both genders

[0191] Sedentary lifestyle (training load less than 2 hours per week in endurance sports)

[0192] Willing to give up any other probiotic products and / or medications known to alter gastrointestinal function during the trial

[0193] The exclusion criteria were:

[0194] Abdominal surgery that could affect GI function (excluding appendectomy and cholecystectomy) as judged by the investigator

[0195] History of peptic ulcer

[0196] Any known bleeding complaints

[0197] Allergy to aspirin

[0198] History of Helicobacter pylori

[0199] Resting diastolic blood pressure ≥ 90 mmHg

[0200] Resting systolic blood pressure ≥140 mmHg

[0201] Current diagnosis of mental illness

[0202] Use of systemic antibiotics, steroids (excluding birth control pills), or antimicrobial drugs in the last 2 months

[0203] BMI>27

[0204] Daily use of nonsteroidal anti-inflammatory drugs in the last 2 months or inadvertent use in the last 2 weeks before screening (aspirin, ibuprofen, diclofenac, naproxen, celecoxib, mefenamic acid, etoricoxib, indomethacin)

[0205] Use of medications in the last 2 weeks before screening, excluding birth control pills

[0206] Diagnosis of inflammatory gastrointestinal disease and / or irritable bowel syndrome

[0207] Lactose intolerance

[0208] Any other disease that may interfere with the subject's intestinal barrier function as judged by the investigator Participation in other clinical trials in the past 2 months before screening

[0209] Regular use of probiotics in the last 2 months

[0210] Smoking and / or frequent use of other nicotine products

[0211] Expect and / or plan to change your current diet and / or exercise regimen during participation in this trial

[0212] Use of laxatives, antidiarrheals, anticholinergics, and PPIs in the last 2 months prior to screening

[0213] Use of immunosuppressant medications within the last 4 weeks prior to screening

[0214] For women: pregnancy or lactation.

[0215] The primary endpoint was the effect of 8 weeks of oral supplementation with DSM 32356 compared with placebo on small intestinal mucosal damage measured as the area under the curve of the Lewis score on capsule endoscopy between Visit 2 (randomization) and Visit 7 (end of treatment) when co-administered to a 6-week acetylsalicylic acid (aspirin challenge).

[0216] The first of the ordered secondary endpoints was the effect of 8 weeks of oral supplementation with DSM 32356 compared with placebo on small intestinal mucosal damage measured as the area under the curve for the number of ulcers from visit 2 to visit 7 from capsule endoscopy when co-administered to a 6-week acetylsalicylic acid (aspirin) challenge.

[0217] Throughout the trial, subjects were instructed to maintain their accustomed lifestyle with respect to diet, physical activity level, and sleep habits.

[0218] Small intestinal mucosal deterioration was assessed using capsule endoscopy and indirectly using biomarkers in stool and blood samples obtained at visits 2-7.

[0219] Starting from the screening visit and until the end of the intervention period, the intake of probiotic products and foods and food supplements containing probiotics was not allowed. Subjects were not withdrawn from the trial because of a single violation, but violations were recorded as protocol deviations.

[0220] The use of any illicit drugs (stimulants or stimulants, such as marijuana, opium) is prohibited during the trial.

[0221] Subjects are required to:

[0222] Avoiding consumption of foods that may contain viable microorganisms (ie, fermented dairy products)

[0223] Avoid taking any nonsteroidal anti-inflammatory drugs (NSAIDs) during the trial period

[0224] Avoid participating in other clinical trials

[0225] Avoid drinking alcohol 2-7 days before your visit

[0226] Avoid spicy foods 2-7 days before the consultation

[0227] Avoid caffeine consumption 2-7 days before your appointment

[0228] Avoid strenuous exercise 2-7 days before the visit

[0229] Fast overnight from (10:00) prior to attending visit 2-7. Clear fluids such as water, soft drinks, decaffeinated tea / coffee are allowed until midnight the day before the visit.

[0230] Capsule endoscopy is used to detect small bowel inflammation and damage in the form of villous edema, ulcers, and strictures. During visits 2-7, the capsule endoscopy procedure was used using the Pillcam capsule from Medtronic.

[0231] Subjects were asked to fast from 10:00 the day before the consultation in order to empty the intestines before the capsule endoscopy procedure. On the morning of the consultation, subjects met the fasting requirement. Before swallowing the Pillcam capsule with a glass of water, the Pillcam signal receiver was connected to the subject's upper body. Then, the Pillcam capsule moved through the body cavity and the small intestine and sent the data in the form of photos to the receiver. The data from the capsule was captured for a total of 8 hours, after which it was expected that the capsule had passed through the small intestine. During the 8-hour capsule recording period, subjects were allowed to leave the scene, but it was recommended that no physical activity be performed during these 8 hours. After 4 hours, subjects were allowed to eat light meals.

[0232] A total of 5 well-trained gastroenterologists participated in the review and scoring of the video data from the capsule. The damage observed in the small intestine from the capsule was divided into three categories: villous edema, ulcers and strictures. These three categories were evaluated separately and also combined into one score for the primary endpoint, the Lewis score (Gralnek et al., 2008) (Gralnek IM, Defranchis R, Seidman E, Leighton JA, Legnani P, Lewis BS. Development of a capsule endoscopyscoring 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 recognized clinical and scientific score for intestinal damage.

[0233] The following rules are used to evaluate data from capsules:

[0234] Security:

[0235] Within one week from the capsule visit, at least one capsule reviewer confirmed that the capsule had passed through the small intestine (by confirming from the last photograph that the capsule was in the colon).

[0236] Data Quality:

[0237] A randomization system was created so that while randomized to the Bifidobacterium breve DSM 32356 / placebo treatment, a subject was also randomized to 2 of 5 reviewers to oversee the capsule data from that subject.

[0238] Two reviewers designated by the randomization system reviewed all capsule data from all capsule consultations for the subject. The two reviewers who oversaw the capsule data from the subject were also blinded to each other and were not allowed to communicate with each other in any way regarding the capsule data.

[0239] The average capsule data value was calculated for those 2 reviewers. In the event that the data from the two reviewers differed by the number of 4 or more ulcers, a third reviewer (the principal investigator) also reviewed the Pillcam data set for that visit and the average for all 3 reviewers was calculated and used as the final data point for that visit.

[0240] The results are presented in graphs showing data from the ITT population.

[0241] As the results show, the trial met its primary endpoint and the first secondary endpoint, showing a clear and statistically significant protective effect of the DSM 32356 strain against aspirin-induced intestinal damage, as observed for the Lewis score (Figure 5), and the total number of ulcers (Figure 6).

[0242] Example 5

[0243] Quantification of Prostaglandin E2 and Thromboxane B2 Concentrations in Blood Samples

[0244] method

[0245] As endpoints, the post-intervention effects on prostaglandin E2 (PGE2) and thromboxane B2 (TBX2), downstream of cyclooxygenase (COX), were investigated in serum samples.

[0246] After unblinding, 2 kinds of biological lipids PGE2 and TXB2 from COX enzyme downstream in fasting serum samples obtained during consultation 2 to consultation 7 were analyzed in the laboratory afterwards. As previously described, extraction scheme and LC-MS / MS analysis (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) were performed by Ambiotis SAS (France). For intervention during analysis, Ambiotis remained blind and analyzed in a single measurement.

[0247] result

[0248] The intake of acetylsalicylic acid (aspirin) was associated with a strong suppression of serum PGE2 and TXB2 concentrations (Figure 7). Bifidobacterium breve DSM 32356 intervention did not alter these well-described acetylsalicylic acid-induced changes in COX downstream metabolites. This suggests that the protective effect of Bifidobacterium breve DSM 32356 does not interfere with the specific cardiovascular protective properties of acetylsalicylic acid.

[0249] Example 6

[0250] Systemic cytokine analysis

[0251] method

[0252] Cytokine Quantification from Heparinized Plasma

[0253] Heparin plasma samples from visit 2 to visit 7 of 31 subjects from the placebo group and 35 subjects from the active group (Bifidobacterium breve, consumers of DSM 32356) were collected to analyze their cytokine profiles. Using the V-plex human proinflammatory response plate 1 from Meso Scale Discovery (Article No. K15049D), according to the manufacturer's protocol, the secretion 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 V-plex single test (Article No. K151STD) according to the manufacturer's guidance, C-reactive protein (CRP) levels were also quantified. Bridge control samples were used to evaluate changes between plates.

[0254] Statistical analysis

[0255] Statistical analysis was performed using GraphPad Prism 7 software (GraphPad Softwar, La Jolla, USA). Cytokine values ​​were compared using a 2-factor ANOVA with Sidak comparison. Data are expressed as median + 95% CI in mg / ml for CRP and in pg / ml for the remaining cytokines. When a value was below the limit of detection or below the fitted curve, it was replaced by half of the lower limit of detection (LOD).

[0256] result

[0257] 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 measurements of IFN-γ, IL-10, IL-6, IL-8, TNF-α, and CRP were within the detection range, and their inter-plate variation values ​​were less than 20% (see Tables 3 and Figures 8 to 13 For IL-12p70, IL-13, IL-1β, IL-2, and IL-4, more than 31.9% of the measurements were below the limit of detection, which increased the inter-plate variation (see Table 3).

[0258] Table 3. Mean values ​​of lower limits of detection for analyzed cytokines, inter-plate variation based on CV values ​​for bridge control samples and % of samples below the limit of detection *Samples measured for IL-13 were 26 for placebo group and 28 for active group.

[0259] Table 3

[0260]

[0261] Example 7

[0262] Bifidobacterium breve recovery and microbiome composition of stool samples

[0263] As endpoints, the post-intervention effects on Bifidobacterium breve abundance and microbiota composition in stool samples were investigated.

[0264] After unblinding, the DNA extracted from all obtained fecal samples using NucleoSpin 96Soil kit (Macherey-Nagel) and randomly sheared into 350bp fragments was subjected to laboratory and bioinformatics analysis afterwards. The library was constructed using the NEBNext Ultra Library Prep kit (New England Biolabs) for Illumina and each sample was sequenced as at least 30 million read pairs (2 × 150bp paired end Illumina sequencing). Sequencing readings were filtered to remove human and low-quality readings, mapped to the clinical microbiome human intestinal 22M gene catalog, and summarized as taxonomic relative abundance tables (Nielsen et al. 2014) Nielsen, HB et al. Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes. Nat. Biotechnol. 32, 822-828 (2014). For intervention during analysis, participants remained blinded. The Wilcoxon rank sum test was used to detect changes in the relative abundance of categories between the scores of visit 2 and later time points and the Bonferroni correction was used to correct for multiple comparisons. Similarly, the Bray-Curtis distances between visit 2 and later time points were compared between the two arms (t-test).

[0265] result

[0266] DNA sequencing of all stool samples obtained showed a clear increase in the abundance of Bifidobacterium breve after randomization in stool samples obtained from subjects in the Bifidobacterium breve DSM32356 arm compared to the placebo arm, confirming compliance with the study product ( Fig.14 ).

[0267] Intervention with Bifidobacterium breve DSM 32356 was not associated with significant changes in the abundance of specific microbial taxa, nor with changes in the overall microbiome composition as revealed by the Bray-Curtis dissimilarity index ( Fig.15 ).

[0268] Example 8

[0269] The overall goal of this experiment was to explore whether Bifidobacterium breve DSM 32356 affects the degradation of aspirin in vitro.

[0270] Assumptions

[0271] In the clinical trial, each subject was administered 300 mg of aspirin (acetylsalicylic acid) per day. Assuming this dose is taken with about 1 dL of water, it would correspond to 0.3 g / 0.1 L = 3 g / L. The molecular weight of aspirin is 180.157 g / mol. The hypothetical maximum exposure in the intestine would be (3 g / L) / (180.157 g / mol) = 0.01665 mol / L = 16.65 mM.

[0272] In order to cover a wide concentration range of acetylsalicylic acid, the following concentrations were used: 20 mM, 10 mM, 5 mM, 2.5 mM, 1.25 mM, 0.625 mM, 0.3125 mM and 0 mM of acetylsalicylic acid.

[0273] Preparation of Bifidobacterium breve

[0274] Two days before incubation with acetylsalicylic acid, Bifidobacterium breve DSM 32356 was cultured anaerobically overnight in Man Rogosa Sharp (MRS) broth (BD Difco 288110, UK) with 0.05% cysteine ​​hydrochloride (CyHCl) (Merck 102839, Germany). The next day, the growing culture was re-inoculated in fresh MRS with 0.05% CyHCl (100 μL bacterial suspension in 10 mL MRS with 0.05% CyHCl). Six dilution rows were produced by transferring 1 ml of the mixed inoculum 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 acetylsalicylic acid, the optical density (OD 600nm) Evaluate bacterial growth and select cultures representing late exponential / early stationary phase. For every 2 dilution rows, combine 2 vials representing late exponential / early stationary phase (add up to 3 vials in total) and, together with the culture medium control vial, centrifuge the tube at 3500×g for 5 minutes to collect the bacterial pellet. Discard the supernatant and add 35ml of MRS with 0.05% CyHCl and rinse the bacteria and centrifuge at 3500×g for 5 minutes. Repeat the rinse procedure once. Further, add 15ml of MRS with 0.05% to each of the 3 vials with bacterial pellets. After resuspending the pellet, combine 3×15mL bacterial suspensions in 1 tube and centrifuge at 3500×g for 10 minutes and discard the supernatant. Add a total of 20mL of MRS with 0.05% CyHCl and resuspend the bacteria. OD 600nm Adjusted to 12, corresponding to about 4.25x10 9 Bacterial density in CFU / mL.

[0275] Preparation of acetylsalicylic acid solution

[0276] Weigh 400mg acetylsalicylic acid, Sigma, A6810 and dissolve in 100ml MRS with 0.05% CyHCl. Use 2M NaOH (diluted from 27% sodium hydroxide solution, Merck) to adjust pH to the pH of culture medium control (pH=6). Aspirin solution is diluted 2 times 6 times in MRS with 0.05% CyHCl to obtain aspirin 22.2mM, 11.1mM, 5.6mM, 2.8mM, 1.39mM, 0.69mM and 0.35mM at the following concentrations. All solutions, including the culture medium control of MRS with 0.05% CyHCl, are sterile filtered.

[0277] Incubation of Bifidobacterium breve DSM 32356 with aspirin

[0278] 9 ml of each acetylsalicylic acid solution (22.2 mM, 11.1 mM, 5.6 mM, 2.8 mM, 1.39 mM, 0.69 mM and 0.35 mM) and control condition (0 mM) were added to different sterile tubes in duplicate. To one dilution row, 1 ml of DSM32356 suspension was added to each tube to achieve final concentrations of acetylsalicylic acid of 20 mM, 10 mM, 5 mM, 2.5 mM, 1.25 mM, 0.625 mM, 0.3125 mM and 0 mM and 4.25×10 8CFU / mL of bacteria. To the second dilution row, 1 ml of MRS with 0.05% CyHCl was added to each tube and the pH was adjusted to 4.5 using approximately 30 μL of HCl (sodium chloride, VWR Chemicals). Both dilution rows were incubated anaerobically at 37°C for 24 hours. After incubation, the pH in the vials was measured and the solutions were sterile filtered and frozen at -80°C until metabolite analysis.

[0279] Metabolite analysis

[0280] All samples were diluted 10 times in 10 mM ammonium formate (from VWR Chemicals, LC-MS grade) with 0.1% formic acid (from VWR Chemicals, LC-MS grade). To ensure proper quantification of acetylsalicylic acid and salicylic acid, sample A6 and sample B6 were diluted 2 additional times, sample A7 and sample B7 were diluted 4 additional times and sample A8 and sample B8 were diluted 10 additional times.

[0281] For quality control, a pooled sample (QC sample) was generated by taking a small aliquot from all samples. The QC sample was analyzed at regular intervals throughout the sequence.

[0282] The analysis was performed using a UPLC system (Vanquish, Thermo Fischer Scientific) coupled to a high-resolution quadrupole-orbitrap mass spectrometer (Q ExactiveTM HF hybrid quadrupole-orbitrap, ThermoFischer Scientific). An electrospray ionization interface was used as the ionization source. The analysis was performed in negative and positive ionization modes. QC samples were analyzed in MS / MS mode for identification of compounds. UPLC was performed using a slightly modified version of the protocol described by Doneanu et al. 2011 (Doneanu, CE, Chen, W. & Masseo, JR (2011) Waters Application note, 720004042en).

[0283] Data were processed using Compound Discoverer 3.0 (Thermo Fischer Scientific).

[0284] Compound extraction is performed by extracting all features from the raw data followed by feature detection by grouping features belonging to the same compound. The isotope pattern is used together with the accurate mass to determine the molecular formula. Compound identification is performed by one of three levels of annotation:

[0285] 1) Confident identification by accurate mass, MS / MS spectrum and known retention time from standard identification.

[0286] 2) Annotation is based on two kinds of information and is divided into two sub-levels: a) based on accurate mass and known retention time, as obtained from standards analyzed on the same system, b) based on accurate mass and MS / MS spectra from external libraries.

[0287] 3) Annotation at this level is based on library searches using individual exact quantities and elemental compositions. Library searches were performed with an acceptable mass deviation of + / - 3 ppm.

[0288] Compounds related to the amount of added aspirin were found by calculating the correlation coefficient between the peak areas obtained and the acetylsalicylic acid concentration. The behavior of compounds with correlation coefficients higher than 0.9 was analyzed in more detail to investigate whether they could be acetylsalicylic acid derived metabolites.

[0289] result

[0290] All pH adjusted samples (B1-B8) had similar pH before and after 24 hours incubation, whereas samples inoculated with Bifidobacterium breve DSM 32356 all had a pH of 6 before incubation and a pH of 4-4.5 after incubation (see Table 4).

[0291] Table 4

[0292]

[0293] In all samples containing acetylsalicylic acid, most of the acetylsalicylic acid was converted to salicylic acid after 24 hours of incubation or acid adjustment with Bifidobacterium breve DSM 32356. When comparing the inoculated dilution rows with the acid adjusted dilution rows, no significant differences were observed between the concentrations of acetylsalicylic acid and salicylic acid in the two groups of samples ( Fig.16 ).

[0294] Importantly, the concentrations of the two active compounds, aspirin and salicylic acid, responsible for the therapeutic effect of aspirin, did not differ between the inoculated and acid-adjusted dilution rows. When comparing the added acetylsalicylic acid with the detected peak areas, several compounds seemed to correlate and had increased peak areas when more acetylsalicylic acid was present. However, in the inoculated samples, the bacteria seemed to consume a large amount of compounds present in the culture medium (which is the ability to inhibit bacterial growth when increasing concentrations of aspirin are added). This inhibited growth may be the underlying reason for the observed correlation, which is supported by the fact that these compounds are present in the same amounts in the samples with high aspirin in both the A and B sample groups. In summary, Bifidobacterium breve DSM 32356 showed no degradation of acetylsalicylic acid.

Claims

1. The strain Bifidobacterium breve deposited as DSM 32356.

2. A composition comprising the strain according to claim 1.

3. The composition according to claim 2, further comprising a cryoprotectant.

4. A composition according to claim 2 or 3, wherein the composition comprises at least one other bacterial strain.

5. The composition according to claim 4, wherein the at least one other bacterial strain is Bifidobacterium infantis.

6. The composition according to any one of claims 2-3 and 5, wherein the composition further comprises at least one compound.

7. The composition according to claim 6, wherein the compound is a vitamin, a fiber or another compound selected from fructooligosaccharides (FOS), galacto-oligosaccharides (GOS) or human milk oligosaccharides (HMO).

8. The composition according to any one of claims 2-3 and 5, wherein the composition further comprises a prebiotic.

9. A composition according to any one of claims 2-3, wherein the composition comprises as the only probiotic component the strain Bifidobacterium breve deposited as DSM 32356.

10. A composition according to claim 4, wherein the Bifidobacterium breve strain deposited as DSM 32356 and at least one other strain are present in an amount of at least 1×10 6 The concentration of CFU / g is present.

11. A composition according to claim 5, wherein the Bifidobacterium breve strain deposited as DSM 32356 and at least one other strain are present in an amount of at least 1×10 6 The concentration of CFU / g is present.

12. A composition according to claim 4, wherein the Bifidobacterium breve strain deposited as DSM 32356 and at least one other strain are present in an amount of at least 1×10 6 CFU / day.

13. A composition according to claim 5, wherein the Bifidobacterium breve strain deposited as DSM 32356 and at least one other strain are present in an amount of at least 1×10 6 CFU / day.

14. Use of Bifidobacterium breve deposited as DSM 32356 for the preparation of a medicament for supporting defense against intestinal tissue damage caused by nonsteroidal anti-inflammatory drugs (NSAIDs) in a subject in need thereof.

15. The use according to claim 14, wherein the medicament is used during administration of an NSAID.

16. The use according to claim 14 or 15, wherein the medicament is used during administration of acetylsalicylic acid.

17. The use according to claim 14 or 15, wherein the medicament is used to reduce intestinal mucosal rupture or damage.

18. A method for producing a feed product or a food product, a dietary supplement or a pharmaceutical composition, comprising producing a bacterium of the strain Bifidobacterium breve deposited under the number DSM 32356 according to claim 1, and 6 The bacteria are added to a feed product or food product, a dietary supplement or a pharmaceutical composition at a concentration of CFU / g.

Images