Probiotic bifidobacterium breve for use in prevention of extra-intestinal symptoms of intestinal bowel syndrome

Abstract

The present invention relates to Bifidobacterium breve or Bifidobacterium breve strain deposited as DSM 32356 or compositions comprising said bacteria for use in preventing, reducing, or ameliorating fatigue symptoms of Intestinal Bowel Syndrome, in particular diarrhea predominant Intestinal Bowel Syndrome.

Description

[0001] TITLE

[0002] Probiotic Bifidobacterium breve for use in prevention of extra-intestinal symptoms of Intestinal Bowel Syndrome

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to Bifidobacterium breve and Bifidobacterium breve deposited as DSM 32356 for use in preventing, reducing, or ameliorating fatigue symptoms of Intestinal Bowel Syndrome or diarrhea-predominant Intestinal Bowel Syndrome. Further the invention relates to compositions comprising the bacterium.

[0005] BACKGROUND ART

[0006] Irritable bowel syndrome (IBS) is a symptom-based disorder of gut-brain interactions generating abdominal pain. The worldwide prevalence of IBS in adults was evaluated to be between 4-9 % with diarrhea-predominant Intestinal Bowel Syndrome (IBS-D) as the most common subtype amounting to 31.5% (Priya Oka, Heather Parr, Brigida Barberio, Christopher J. Black, Edoardo V. Savarino, Alexander C. Ford (2020) Global prevalence of irritable bowel syndrome according to Rome III or IV criteria: a systematic review and meta-analysis. Lancet Gastroenterol Hepatol. Oct;5(10):908-917). According to the Rome IV diagnostic criteria (Max J Schmulson and Douglas A Drossman (2017) What Is New in Rome IV. J Neurogastroenterol Motil. Apr 30;23(2): 151-163) IBS is defined as a functional bowel disorder in which patients report recurrent abdominal pain on an average at least one day in a week in the last 3 months, associated with two or more symptoms such as pain during defecation, change in stool frequency and stool form. IBS-D is defined as; more than one-fourth (25%) of bowel movements with Bristol Stool Scale Types 6-7 and less than one-fourth (25%) with Types 1-2. Non-gastrointestinal (GI) symptoms such as depression, anxiety and fatigue can occur in combination with GI symptoms in IBS (Han CJ and Yang GS, Fatigue in Irritable Bowel Syndrome (2016). A Systematic Review and Meta-analysis of Pooled Frequency and Severity of Fatigue. Asian Nurs Res (Korean Soc Nurs Sci), Mar; 10(1): 1-10). In addition, fatigue has been reported as one of the most common and disturbing symptoms of these extra-intestinal symptoms (Anna-Karin Norlin, Susanna Walter, Adriane Icenhour, Asa V. Keita, Sigrid Elsenbruch, Olga Bednarska, Michael P. Jones 5, Rozalyn Simon, Maria Engstrom (2021). Fatigue in irritable bowel syndrome is associated with plasma levels of TNF-a and mesocorticolimbic connectivity. Brain Behav Immun, Feb:92:211-222). Results of a recent study with fecal microbiota transplantation (FMT) recently indicated effect of FMT on fatigue in IBS (Johnsen PH, Hilpiisch F, Valle PC, Goll R (2020). The effect of fecal microbiota transplantation on IBS related quality of life and fatigue in moderate to severe non-constipated irritable bowel: Secondary endpoints of a double blind, randomized, placebo-controlled trial. EBioMedicine, Jan;51 : 102562).

[0007] When fatigue is associated with irritable bowel syndrome (IBS), the treatment approach may involve addressing both the gastrointestinal symptoms of IBS and the fatigue itself. Treatment strategies for fatigue deriving from IBS may involve a combination of lifestyle modifications, dietary changes, and, in some cases, medications.

[0008] IBS patients often have a lower level of Bifidobacteria and Lactobacilli in their microbiome compared to healthy controls. Attempts have therefore made to treat patients with IBS and IBS-associated symptoms such as abdominal pain, bloating and flatulence with prebiotics such as fructooligossacharides (FOS) and galactooligosaccharides (GOS) and probiotics such as Bifidobacteria and Lactobacilli. However, because only limited data are available and because of the heterogeneity of these studies, the use of prebiotics and probiotics for treatment of IBS and IBS related symptoms, said treatments with probiotics and prebiotics have not been generally accepted by the Gastroenterology community (Sofia D Shaikh, Natalie Sun, Andrew Canakis, William Y Park and Horst Christian Weber (2023) Irritable Bowel Syndrome and the Gut Microbiome: A Comprehensive Review, J Clin Med. 2023 Mar 28;12(7):2558).

[0009] Thus, there is a desire to provide a solution for preventing, reducing, and / or ameliorating of symptoms associated with IBS including IBS associated fatigue.

[0010] SUMMARY OF THE INVENTION

[0011] An objective of the present invention is to address the above-mentioned drawbacks, in particular to obtain probiotic bacteria which can prevent, reduce, and / or ameliorate fatigue symptoms of IBS. According to a first aspect, this and further objects are achieved by the provision of Bifidobacterium breve or Bifidobacterium breve strain deposited as DSM 32356 for use in preventing, reducing, and / or ameliorating fatigue symptoms of Intestinal Bowel Syndrome (IBS) in a subject in need thereof.

[0012] The Bifidobacterium breve strain deposited as DSM 32356 is also denoted Bifl95.

[0013] In a preferred embodiment the IBS is diarrhea-predominant Intestinal Bowel Syndrome (IBS-D).

[0014] The Bifidobacterium breve or Bifidobacterium breve strain (DSM 32356) may be provided as a live, dead or fragmented culture or in a composition which may be freeze-dried and / or provided in a capsule.

[0015] In another embodiment, the culture and / or composition comprises at least one other bacterial strain. In one embodiment the culture or composition comprises Bifidobacterium breve or the Bifidobacterium breve strain (DSM 32356) as the only probiotic component.

[0016] The Bifidobacterium breve or the specific Bifidobacterium breve strain (DSM 32356) may be administered in an amount of at least 1 x 107CFU / day, such as 107CFU / day, 108CFU / day, 109CFU / day or IO10CFU / day. In one embodiment the Bifidobacterium breve strain (DSM 32356) may administered in an amount in the range of 1 x 108CFU / day to 1 x IO10CFU / day, such as 15*109CFU / day or 46*109CFU / day or 50 *109CFU / day.

[0017] In another aspect of the invention, the above objectives are achieved by providing a method of preventing, reducing, and / or ameliorating fatigue symptoms of Intestinal Bowel Syndrome according, the method comprising administering the Bifidobacterium breve strain to a subject in need thereof.

[0018] The invention also relates to a method of preventing, reducing, and / or ameliorating fatigue symptoms of Intestinal Bowel Syndrome in a subject in need thereof, the method comprising administering Bifidobacterium breve or Bifidobacterium breve strain deposited as DSM 32356 to said subject in need thereof as described herein.

[0019] Bifidobacterium breve or the Bifidobacterium breve strain (DSM 32356) may be administered to the patient in the form of a feed or food product, dietary supplement or pharmaceutical composition comprising said Bifidobacterium breve or Bifidobacterium breve strain Bifidobacterium breve (DSM 32356). DETAILED DISCLOSURE OF THE INVENTION

[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by persons 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. The invention may be embodied in many different forms within the scope of the claims set out herewith and should not be contrived as limited to the embodiments as set forth herein; rather, these embodiments are provided for thoroughness and completeness.

[0021] The present invention is based upon the results of a randomized controlled clinical trial where patients diagnosed with diarrhea-predominant Intestinal Bowel Syndrome (IBS-D) were recruited to evaluate the effect of Bifidobacterium breve DSM 32356 with placebo on different symptom outcomes one of them being fatigue.

[0022] Fatigue in IBS may be associated with gastrointestinal symptoms. IBS and IBS-D primarily involves symptoms related to the digestive system. Common symptoms include: abdominal pain or discomfort which typically may be relieved after a bowel movement bloating : a sensation of increased abdominal pressure or fullness altered bowel habits: Changes in frequency and consistency of bowel movements, including diarrhea, constipation, or a mix of both.

[0023] Fatigue in IBS or IBS-D may also be postprandial as it is often associated with meals or after bowel movements. The energy drain may be linked to the body's response to the digestive process and associated symptoms.

[0024] Stress-Related Fatigue: Stress and anxiety can exacerbate IBS symptoms, including fatigue. Stress management is crucial in managing IBS-related fatigue.

[0025] Diarrhea-predominant Irritable Bowel Syndrome (IBS-D) is one of the subtypes of Irritable Bowel Syndrome characterized by recurrent episodes of diarrhea along with other gastrointestinal symptoms. The symptoms associated with IBS-D can vary in severity and may include: frequent diarrhea: individuals with IBS-D experience recurrent episodes of loose or watery stools, often accompanied by an urgent need to have a bowel movement, urgency: there is a sense of urgency to use the bathroom, and the need to evacuate the bowels can be sudden and intense. incomplete bowel movements: despite urgency, individuals may feel that they have not completely emptied their bowels. abdominal discomfort during or after bowel movements: pain or discomfort may be particularly noticeable during or after bouts of diarrhea.

[0026] The study enrolled 61 patients diagnosed with IBS-D according to the ROME-IV criteria (Rome IV Diagnostic Questionnaires and Tables for Investigators and Clinicians," published in 2016 by the Rome Foundation) (Palsson, O. S., Whitehead, W. E., Van Tilburg, M. A. L., Chang, L., Chey, W., Crowell, M. D., Keefer, L., Lembo, A. J., Parkman, H. P., Rao, S. S. C., Sperber, A., Spiegel, B., Tack, J., Vanner, S., Walker, L. S., Whorwell, P., & Yang, Y. (2016). Rome IV Diagnostic Questionnaires and Tables for Investigators and Clinicians. Gastroenterology, 150(6), 1481-1491) with moderate to severe disease activity. The disease activity is defined as IBS Symptom Severity Score (IBS-SSS) > 175 (Francis, C. Y., Morris, J., & Whorwell, P. J. (1997). The irritable bowel severity scoring system: a simple method of monitoring irritable bowel syndrome and its progress. Alimentary Pharmacology & Therapeutics, 11(2), 395-402).

[0027] For 8 weeks participants were supplemented with either capsules of Bifidobacterium breve deposited as DSM 32356 (B. breve DSM 32356) or placebo and followed up for additional 8 weeks with no supplementation.

[0028] Fatigue was measured by the Fatigue Numeric Rating Scale (Fatigue NRS) (Gladman, D., Nash, P., Goto, H., Birt, J. A., Lin, C. Y., Orbai, A. M., & Kvien, T. K. (2020). Original article: Fatigue numeric rating scale validity, discrimination and responder definition in patients with psoriatic arthritis, RMD Open, 6(1)) which was a single-item patient-reported outcome (PRO) where participants evaluate their subjective feeling of fatigue, daily, 7 days prior to starting the intervention and 7 days prior to ending the intervention.

[0029] In accordance herewith, the present invention relates to Bifidobacterium breve deposited as DSM 32356 for use in preventing, reducing, or ameliorating fatigue symptoms of Intestinal Bowel Syndrome, in particular symptoms of diarrhea-predominant Intestinal Bowel Syndrome. BRIEF DESTRIPTION OF DRAWINGS

[0030] Figure 1

[0031] Shows a plot of the fatigue score against week 0 to week 16 for DSM 32356 and Placebo.

[0032] DETAILED DESCRIPTION

[0033] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "having", "including" and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any nonclaimed element as essential to the practice of the invention.

[0034] In the example it is shown that DSM 32356 {Bifidobacterium breve deposited as DSM 32356) reduce fatigue statistically significantly compared to placebo assessed by the Fatigue Numeric Rating Scale (Fatigue NRS) in a clinical study. The present disclosure provides a method of preventing, reducing, or ameliorating fatigue in a subject in need thereof, with Bifidobacterium breve DSM 32356.

[0035] In the context of this invention, the term "probiotic component" refers to a culture of live or freeze-dried microorganisms, dead microorganisms, fragments of microorganisms and extracts or supernatants of microorganisms which, when applied to man or animal, beneficially affects the host (FAO / WHO, 2001).

[0036] DEPOSIT AND EXPERT SOLUTION The present invention provides Bifidobacterium breve deposited according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Inhoffenstr. 7B, D-38124 Braunschweig on August 9th, 2016 under the accession number DSM 32356.

[0037] Table: The applicant has made the following deposits at a Depositary institution having acquired the status of international depositary authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure: Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures Inhoffenstr. 7B, 38124 Braunschweig, Germany.

[0038] The present invention further provides compositions comprising said strain.

[0039] A bacterial "strain" as used herein refers to a bacterium which remains genetically unchanged when grown or multiplied.

[0040] In a further aspect, the invention provides the Bifidobacterium breve strain deposited as DSM 32356 for use in the support of preventing, reducing, or ameliorating fatigue symptoms from Intestinal Bowel Syndrome, in particular diarrhea-predominant Intestinal Bowel Syndrome.

[0041] In the present context, the term "Intestinal Bowel Syndrome" is to be understood as an identified and classified functional gastrointestinal disorder according to the ROME-IV criteria

[0042] In the present context, the term "diarrhea-predominant Intestinal Bowel Syndrome" is to be understood as an identified and classified functional gastrointestinal disorder according to the ROME-IV criteria.

[0043] The invention further relates to a method of for use in preventing, reducing, or ameliorating fatigue symptoms from diarrhea-predominant Intestinal Bowel Syndrome.

[0044] In one embodiment, the compositions comprising Bifidobacterium breve deposited as DSM 32356 as described herein may comprise at least one other bacterial strain and / or at least one compound such as vitamins, prebiotics, fibers or other compounds which may have a beneficial health effect such as fructo-oligosaccharides (FOS), galacto-oligosaccharide (GOS) or human milk oligosaccharides. For example, the compositions of the present invention may comprise bacteria of the Bifidobacterium breve strain deposited as DSM 32356 and at least one other bacterial strain.

[0045] The compositions of the present invention may comprise bacteria of the species Bifidobacterium breve or the strain Bifidobacterium breve deposited as DSM 32356 together with bacteria 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 easel subsp. casei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus 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, or from the genera of Akkermansia, Anaerostipes, Butyricicoccus, Christensenella, Clostridia, Coprococcus, Dorea, Eubacterium, Faecalibacterium or Roseburia or the family Coriobacteriaceae.

[0046] Thus, the composition may further comprise at least one strain of a bacterium selected from the group comprising 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.

[0047] In presently preferred embodiments, only one, two, three, four or five different strains are present in a composition according to the invention. In one aspect of this embodiment the compositions of the present invention comprise bacteria of the species Bifidobacterium breve, for example bacteria of the Bifidobacterium breve strain deposited as DSM 32356, and bacteria of the strain Bifidobacterium animalis subsp. lactis, for example bacteria of the Bifidobacterium animalis subsp. lactis strain deposited as DSM 15954. The composition may comprise bacteria of these two strains as the only probiotic component.

[0048] In another aspect of this embodiment the compositions of the present invention comprise bacteria of the strain Bifidobacterium breve, for example bacteria of the Bifidobacterium breve strain deposited as DSM 32356, and bacteria of the strain Lactobacillus rhamnosus, for example bacteria of the Lactobacillus rhamnosus strain deposited as ATCC 53103. The composition may comprise bacteria of these two strains as the only probiotic component.

[0049] In further aspect of this embodiment the compositions of the present invention comprise bacteria of the strain Bifidobacterium breve, for example bacteria of the Bifidobacterium breve strain deposited as DSM 32356, bacteria of the strain Bifidobacterium animalis subsp. lactis, for example bacteria of the Bifidobacterium animalis subsp. lactis strain deposited as DSM 15954 and bacteria of the strain Lactobacillus rhamnosus, for example bacteria of the Lactobacillus rhamnosus strain deposited as ATCC 53103. The composition may comprise bacteria of these three strains as the only probiotic component.

[0050] In another embodiment of this aspect bacteria of the Bifidobacterium breve strain deposited as DSM 32356 are the only probiotic component in the composition.

[0051] The compositions of the present invention may comprise the bacteria in any suitable form for administration to the subject. In a preferred embodiment, the compositions of the present invention may comprise the bacteria in dried form, which can be obtained by freeze-drying, spray-drying or lyophilization.

[0052] If the bacteria are freeze-dried, they are generally mixed with a cryoprotectant before they are freeze-dried in order to obtain a high viability. The term "a cryoprotectant" is used in the context of the present invention to refer to a substance that is able to improve the survival during freezing and / or drying and to improve the storage stability of bacteria. The cryoprotectant used herein preferably comprises a saccharide.

[0053] The saccharide may be a mono-, di-, oligo- or polysaccharide, or a mixture of at least two saccharides. Useful monosaccharides include glucose (also known as dextrose), fructose, ribose and galactose and useful disaccharides include among other sucrose, trehalose, maltose and lactose. The composition may comprise one or more mono- or disaccharides, such as one, two, or three or even more different saccharides.

[0054] As an example, the cryoprotectant may comprise a mixture of a disaccharide, such as sucrose, and a polysaccharide, such as maltodextrin.

[0055] The cryoprotectant may further comprise a peptide, protein, protein hydrolysate or a mixture thereof. Examples of peptides and proteins to be used are casein, pea, whey, albumin, soy protein, glutamic acid or gelatin, and any isolate or hydrolysate thereof. Other additives, e.g. antioxidants such as ascorbate, sodium citrate, propyl gallate may also be present.

[0056] In one embodiment, the composition of the present invention comprises bacteria of the Bifidobacterium breve strain deposited as DSM 32356 in frozen or freeze-dried form and a cryoprotectant. The cryoprotectant may comprise a saccharide. 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.

[0057] It is preferred that the compositions of the present invention are administered orally. The compositions are thus typically in a form suitable for oral administration. The composition may be a solid or a liquid composition. The composition may be in unit dosage form. For example, the composition can be a capsule, pastille, a pill, a tablet, a soft gel, a sachet, a stick, a stick powder, or in a more general composition such as oil drops, an emulsion, or a paste, or in any other suitable carrier determined by those of skill in the art to be an effective carrier for live organisms.

[0058] The compositions may be encapsulated for example using a suitable polymeric matrix to improve long-term stability and storage of the compositions. Those skilled in the art will appreciate that any suitable encapsulation material or matrix and encapsulation methods and techniques known to those skilled in the art may be used.

[0059] 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 milk product e.g., a yogurt or an infant formula.

[0060] In a further embodiment, the compositions of the present invention comprise bacteria of the Bifidobacterium breve or Bifidobacterium breve strain deposited as DSM 32356 in a unit dosage form, or a more general composition as described above or as part of a dietary supplement or a feed or food product such as a fermented milk product e.g., a yogurt or an infant formula.

[0061] In one embodiment of the present invention, the composition of the present invention may be used as a prophylactic treatment to support the defense against intestinal tissue damage in a subject having an increased risk of intestinal tissue damage. In another embodiment of the present invention, the composition of the present invention may be used as a treatment to support the defense against intestinal tissue damage in a subject having intestinal tissue damage.

[0062] As used herein the terms "treating" and "treatment" refer to all applications which alleviate, remedy, or otherwise hinder, retard, heal, or reverse the progression of, a disease or disorder or at least one symptom of a disease or disorder, including reducing the severity of a disease or disorder. Thus, treatment does not necessarily imply that a subject is treated until complete recovery from a disease or disorder. Similarly, the terms "prophylactic", "preventing", "prevention" and the like refer to any and all applications that prevent intestinal tissue damage in a subject, which may lead to the establishment of a disease or disorder.

[0063] The term "subject” as used herein refers to any mammal, including, but not limited to, livestock and other farm animals (such as cattle, goats, sheep, horses, pigs and chickens), performance animals (such as racehorses), companion animals (such as cats and dogs), laboratory test animals and humans. Typically, the subject is a human.

[0064] In one embodiment, the composition of the present invention comprises bacteria of the Bifidobacterium breve or the Bifidobacterium breve strain deposited as DSM 32356 in dried, frozen or freeze dried form and / or the composition is administered in a concentration of from 1 x 108CFU / day to 1 x 1011CFU / day.

[0065] Those skilled in the art will appreciate that the administration of compositions disclosed herein can be carried out with dose levels and dosing regimens as required depending on the circumstances and on the condition of the subject. Suitable dosage regimes can be determined based on the teaching of the present application. Dosage regimens may be adjusted to provide the optimal support against intestinal tissue damage of the subject. Persons skilled in the art will appreciate that the exact amounts and rates of administration of the Bifidobacterium breve strain will depend on a number of factors such as the age, body weight, general health, sex and dietary requirements of the subject. Based on the teaching herein those skilled in the art will, by routine trial and experimentation, can determine suitable dosage regimes on a case-by-case basis.

[0066] In an exemplary embodiment of the present invention, the composition of the present invention may be administered daily for at least 1 day. Alternatively, the composition can 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 more.

[0067] Accordingly, the present invention provides a composition of the present invention comprising bacteria of the Bifidobacterium breve or the Bifidobacterium breve strain deposited as DSM 32356 in dried, frozen or freeze-dried form wherein the composition is administered in a dose of from 1 x 108CFU / day to 1 x 1011CFU / day for at least 2 weeks.

[0068] In a separate embodiment, the present invention provides a composition of the present invention comprising bacteria of the Bifidobacterium breve strain deposited as DSM 32356 in dried, frozen or freeze dried from wherein the composition is orally administered in a dose of from 1 x 108CFU / day to 1 x 1011CFU / day for at least 8 weeks.

[0069] In a further aspect the present invention provides a combination treatment comprising a composition which comprises Bifidobacterium breve bacteria or the Bifidobacterium breve strain deposited as DSM 32356, and at least one compound as described herein for coadministration to a subject in need thereof. The compositions may be formulated as combined or as separate compositions of the bacterial strain or strains and the compound e.g., the NSAID.

[0070] In a separate embodiment, the Bifidobacterium breve or the Bifidobacterium breve strain deposited as DSM 32356 is incorporated into a feed or food product such as an infant formula, health food, food additive, dietary supplement, pharmaceutical or over-the- counter formulation in a solid form such as a powder, a tablet, or a liquid form.

[0071] In a preferred embodiment, the strain of Bifidobacterium breve or Bifidobacterium breve strain deposited as DSM 32356 and the at least one other strain, if applicable, is / are present in an amount of at least 107CFU per capsule. Specifically, the amount may be at least 1 x 107CFU, at least 1 x 108, 1 x 109CFU, 1 x IO10CFU, 1 x 1011CFU or 1 x 1012CFU per capsule. In a further aspect, the present invention provides a method for producing a feed or food product, dietary supplement or pharmaceutical composition comprising producing bacteria of the Bifidobacterium breve strain deposited under number DSM 32356, and incorporating the same into a food product, dietary supplement, or pharmaceutical composition in a concentration of at least 107CFU / g.

[0072] DEPOSIT and EXPERT SOLUTION

[0073] The applicant requests that a sample of the micro-organism deposited for the present application as described below may only be made available to an expert, until the date on which the patent is granted.

[0074] Bifidobacterium breve was deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on August 9th, 2016 under the accession no. DSM 32356.

[0075] EXAMPLE

[0076] The present example is a clinical study illustrating the ability of DSM 32356 to reduce symptoms fatigue symptoms in patients diagnosed diarrhea-predominant Intestinal Bowel Syndrome.

[0077] This study is a double-blind randomized, placebo-controlled study designed to evaluate the effects of 8 weeks treatment with DSM 32356 in 61 patients with diarrhea-predominant irritable bowel syndrome (IBS-D). Patients will be randomly allocated 1 :1 to DSM 32356or placebo.

[0078] Patients will participate in the study for a total duration of approximately 16 weeks excluding the screening phase. Besides the screening visit, the study will consist of 4 visits at site.

[0079] At the screening visit, clinical history, blood samples for abnormal biochemistry and symptoms will be assessed. Fecal samples were obtained at all study visits. Patients were seen for study visits at baseline, 4, 8 and 16 weeks, where they completed an IBS-severity scoring system (IBS-SSS), IBS-specific quality of life (IBS-QoL) and questionnaire about bowel habits (frequency / day) and Bristol Stool Form Scale. 61 adults diagnosed with IBS-D according to Rome IV criteria were included in the study. The study incorporated an 8-week intervention period (2 arms) with 8-week follow-up:

[0080] Test product: 1 capsule / day of 15 billion CFU Bifidobacterium breve strain deposited as DSM 32356 starting at baseline for a duration of 8 weeks. This strain is also 5 denoted B. breve DSM 32356.

[0081] Reference product (placebo) : 1 placebo capsule identical in appearance and packaging starting at baseline for at duration of 8 weeks.

[0082] Fatigue was assessed at baseline, after 4 weeks, after 8 weeks and again after 16 weeks. 10 Statistical difference in change in fatigue from baseline was assessed after 8 weeks and compared between treatment and placebo.

[0083] The results are shown in Table 1 below and in Figure 1.

[0084] Table 1. The effect of daily intake of DSM 32356vs. placebo on fatigue (FAS)

[0085] DSM 32356 Placebo Overall

[0086] (N=31) (N=30) (N=61) *P-Value

[0087] At baseline (VI) n 31 30 61

[0088] Mean (SD) 4.5 (1.87) 4.6 (2.02) 4.5 (1.93)

[0089] Median 4.6 4.9 4.7

[0090] Min;Max 0.6;7.6 0.4;8.7 0.4;8.7

[0091] Q1;Q3 3.4;6.0 3.1;6.3 3.4;6.1

[0092] After 8 weeks treatment (V3) n 30 30 60

[0093] Mean (SD) 3.7 (2.15) 4.7 (2.22) 4.2 (2.22)

[0094] Median 3.4 4.9 4.6

[0095] Min;Max 0.0;7.4 0.0;10.0 0.0;10.0

[0096] Q1;Q3 2.2;5.1 3.4;5.7 2.7;5.6

[0097] Difference between baseline (VI) and n 30 30 60 0.0139

[0098] (V3)

[0099] Mean (SD) -0.7 (1.78) 0.1 (1.65) -0.3 (1.75)

[0100] Median -0.6 0.1 -0.4

[0101] Min;Max -4.7;2.9 -4.0;2.9 -4.7;2.9 Table 1. The effect of daily intake of DSM 32356vs. placebo on fatigue (FAS)

[0102] DSM 32356 Placebo Overall

[0103] (N=31) (N=30) (N=61) *P-Value

[0104] Q1;Q3 -1.9;0.0 -0.7;1.3 -1.7;0.6

[0105] N = number of subjects within population, n = number of subjects fulfilling criteria, SD = Standard Deviation, Q1,Q3 - Lower Quartile and Upper Quartile, Min, Max - Minimum and Maximum.

[0106] * = ANCOVA analysis of the delta values (V3 - VI), with VI values as covariate and adjusted for age and gender. FAS: Full Analyses Set

[0107] Figure 1 shows fatigue score from week 0 to week 16. Table 1 show fatigue score at baseline (Visit 1, VI) and after 8 weeks of treatment (Visit 3, V3), the delta changes from

[0108] 5 baseline (V3 minus VI) and the analysed statistical difference. SEM is Standard Error of the Mean.

[0109] Inclusion criteria:

[0110] Adult patients with IBS with diarrhea (IBS-D) (aged 18-70 years); fulfill Rome IV diagnostic

[0111] 10 criteria for IBS; moderate-to-severe disease activity (IBS-SSS >175); IBS-D defined as; more than one-fourth (25%) of bowel movements with Bristol Stool Scale Types 6-7 and less than one-fourth (25%) with Types 1-2; able to read and speak Danish; normal colonoscopy (performed within 1 year) if the patient had blood in stool.

[0112] Further details of inclusion criteria:

[0113] 15 1. Diagnosis of IBS-D according to ROME IV criteria

[0114] 2. Moderate-to-severe disease activity (IBS-SSS >175)

[0115] 3. Age 18-70

[0116] 4. Able to read and speak Danish

[0117] 5. Normal colonoscopy (performed within 1 year) if the patient is describing blood in stool

[0118] 20 6. Provided voluntary written informed consent

[0119] Exclusion criteria:

[0120] Other chronic gastro intestinal diseases including lactose intolerance and coeliac disease; fecal calprotectin >50 mg / kg; fecal sample positive for enteropathogenic microorganisms;

[0121] 25 Surgical interventions in the GI region (except for appendectomy, hernia repair and gynecological and urological procedures); severe psychiatric disorder, or not well treated psychiatric disorder, or abuse of alcohol or drugs; medications except birth control pills, hormone supplements, allergies / asthma agents, blood pressure and cholesterol-lowering agents, proton pump inhibitors, non-prescription medicines and psychiatric medicine (if the patient is well treated, treatment has been stable for at least 3 months and it is assessed by a physician not to be the cause of the IBS symptoms); abnormal colonoscopy findings; pregnant, planned pregnancy or breastfeeding females; ingestion of probiotics or antibiotics <4 weeks before the inclusion; abnormal screening biochemistry.

[0122] Further details of exclusion criteria:

[0123] 1. History or diagnosis of another chronic GI disease, including malabsorption diseases such as lactose intolerance and coeliac disease

[0124] 2. Fecal analysis positive for pathogenic microorganisms including Clostridium difficile

[0125] 3. Fecal calprotectin >50mg / kg

[0126] 4. Oral antibiotics or probiotics within 4 weeks prior to the screening visit

[0127] Surgical interventions in the GI region (except for appendectomy, hernia repair and gynecological and urological procedures)

[0128] 6. Severe psychiatric disorder or not well treated psychiatric disorder

[0129] 7. Abuse of alcohol or drugs

[0130] 8. Medications except birth control pills, hormone supplements, allergies / asthma agents, blood pressure and cholesterol-lowering agents, proton pump inhibitors, non-prescription medicines and psychiatric medicines (if the patient is well treated, treatment has been stable for at least 3 months, and it is assessed by a physician not to be the cause of the IBS symptoms)

[0131] 9. Abnormal screening biochemistry

[0132] 10. Pregnant, planned pregnancy or breastfeeding females

[0133] Description of the product and administration of the product:

[0134] Bifidobacterium is a genus of lactic acid- and acetic acid-producing, gram-positive, nonspore forming, non-motile, anaerobic bacteria. They are common constitutes of the microbiota in the human intestinal tract.

[0135] The investigational product is a vegetable capsule containing Bifidobacterium breve DSM 32356.

[0136] The reference product (placebo) is the same capsule, but without DSM 32356.

[0137] The CFU stability of the production batch of Bifidobacterium breve DSM 32356 that will be used in this study will be monitored closely during the study. The internal analyses indicate that the specific product stability loss is approximately 25% during the product shelf life, and we expect that the CFU per capsule will be minimum 46*109CFU per capsule at the time of study intervention. The CFU stability of the investigational product batch will be analyzed in parallel with performing this study. All patients will be given 1 capsule daily with B. breve DSM 32356 starting at baseline for a duration of 8 weeks.

[0138] A dosage of approximately 46*109CFU of DSM 32356 were administered per day in the active treatment part of the study.

[0139] Table 2 Composition of investigational product

[0140] Reference Product (placebo): Placebo capsules are identical with B. breve DSM 32356 in appearance and packaging and will be given 1 / day starting at baseline for a duration of 8 weeks.

[0141] Duration of Intervention:

[0142] 8 weeks intervention and 8 weeks follow-up.

[0143] Written, informed consent was obtained from all subjects prior to entry into the study. Blinding

[0144] Both active (DSM 32356) and reference (placebo) product will be similar in smell, taste and appearance. All study products will be packaged in identical packs with identical labelling, except for the randomization number.

[0145] Patients, the clinical team and the investigator will all be blinded during the entire study.

[0146] The Primary endpoints

[0147] The effects of daily intake of DSM 32356 versus placebo on IBS symptoms (measured by IBS- severity scoring system (IBS-SSS)) after 8 weeks treatment (IBS-SSS is attached as Appendix C)

[0148] The secondary endpoints

[0149] The effects of daily intake of DSM 32356 versus placebo on Irritable Bowel Syndrome - Quality of Life (IBS-QoL) after 8 weeks treatment (IBS-QoL is attached as Appendix D)

[0150] The effects of daily intake of DSM 32356 versus placebo on microbiota composition after 8 weeks treatment

[0151] The effects of daily intake of DSM 32356 versus placebo on abdominal pain, fatigue and change in bowel habits measured by frequency / day and Bristol Stool Form Scale

[0152] The effects of daily DSM 32356 versus placebo on changes in blood markers on antiinflammatory activity and intestinal barrier function after 8 weeks treatment

[0153] The effects of daily intake of DSM 32356 versus placebo on adverse events

[0154] Safety:

[0155] In Europe, strains of Bifidobacterium belonging to the species B. breve have been granted QPS status by EFSA. This means that the strain used in this study is considered safe to use in food and as a dietary supplement.

[0156] Adverse Events (AEs)

[0157] AEs was recorded from Visit 1 until the end of the study at Visit 4. Patients was asked during all visits to site to report any AEs experienced throughout the study. Adverse events were recorded and assessed.

[0158] Sample Handling

[0159] A screening blood sample will be collected and analyzed at the screening visit. Blood samples for the biobank will be collected at visit 1 and visit 3. At each visit, a maximum of 40 mL whole blood will be taken. All blood and fecal samples were stored at the clinical site during the study. Storage condition for these samples is -80°C.

[0160] All samples were analyzed using validated assays and methods. The screening blood sample included the following parameters; Thrombocytes;B, Creatinin (enz.);P, Hemoglobin;B, C-reactive protein [CRP];P, Albumin;P and Alanine transaminase [ALAT];P. Blood samples for the biobank was analyzed for changes in concentrations of i.a. inflammatory markers including interleukins, tumor necrosis factor-o (TNFo) and intestinal permeability markers i.a. zonulin.

[0161] Result and conclusion

[0162] The results of this trial show that DSM 32356 reduce fatigue statistically significantly compared to placebo assessed by the Fatigue Numeric Rating Scale (Fatigue NRS). The present disclosure thus provides a method of preventing, reducing, or ameliorating fatigue in a subject in need thereof, with Bifidobacterium breve DSM 32356.

Claims

CLAIMS1. Bifidobacterium breve for use in preventing, reducing, and / or ameliorating fatigue symptoms of Intestinal Bowel Syndrome in a subject in need thereof.

2. Bifidobacterium breve for use in preventing, reducing, and / or ameliorating fatigue symptoms of Intestinal Bowel Syndrome in a subject in need thereof according to claim 1, wherein the Bifidobacterium breve is the Bifidobacterium breve strain deposited as DSM 32356.

3. Bifidobacterium breve Bifidobacterium breve strain deposited as DSM 32356 for use in preventing, reducing, and / or ameliorating fatigue symptoms of Intestinal Bowel Syndrome in a subject in need thereof according to claims 1 or 2; wherein the Intestinal Bowel Syndrome is diarrhea-predominant Intestinal Bowel Syndrome (D-IBS).

4. Bifidobacterium breve or Bifidobacterium breve strain deposited as DSM 32356 for use in preventing, reducing, and / or ameliorating fatigue symptoms of Intestinal Bowel Syndrome or D-IBS according to any one of claims 1 to 3, wherein the Bifidobacterium breve or Bifidobacterium breve strain deposited as DSM 32356 is administered in an amount of at least 1 x 107CFU / day.

5. Bifidobacterium breve or Bifidobacterium breve strain deposited as DSM 32356 for use in preventing, reducing, and / or ameliorating fatigue symptoms of Intestinal Bowel Syndrome or D-IBS according to claim 4, wherein the Bifidobacterium breve or Bifidobacterium breve strain deposited as DSM 32356 is administered in an amount in the range of 1 x 108CFU / day to 1 x IO10CFU / day.

6. Bifidobacterium breve or Bifidobacterium breve strain deposited as DSM 32356 for use in preventing, reducing, and / or ameliorating fatigue symptoms of Intestinal Bowel Syndrome or D-IBS according to claims 4 to 5, wherein the Bifidobacterium breve or Bifidobacterium breve strain deposited as DSM 32356 is administered in an amount 46 x 109CFU / day.

7. A composition comprising the Bifidobacterium breve or Bifidobacterium breve strain deposited as DSM 32356 for use in preventing, reducing, and / or ameliorating fatigue symptoms of Intestinal Bowel Syndrome or D-IBS in a subject in need thereof according to any one of claims 1 to 6.

8. The composition for use in preventing, reducing, and / or ameliorating fatigue symptoms of diarrhea-predominant Intestinal Bowel Syndrome or D-IBS in a subject in need thereof according to claim 7, wherein the composition comprises at least one other bacterial strain.

9. The composition for use in preventing, reducing, and / or ameliorating fatigue symptoms of Intestinal Bowel Syndrome or D-IBS in a subject in need thereof according to any one of claim 7 to 8 wherein the composition comprises the Bifidobacterium breve or Bifidobacterium breve strain deposited as DSM 32356 as the only probiotic component.

10. A capsule comprising Bifidobacterium breve or Bifidobacterium breve strain deposited as DSM 32356 or a composition thereof for use in preventing, reducing, and / or ameliorating fatigue symptoms of Intestinal Bowel Syndrome or D-IBS in a subject in need thereof according to any one of claims 1 to 9, wherein the Bifidobacterium breve or Bifidobacterium breve strain deposited as DSM 32356 is present in an amount of at least 1 x 107CFU.

11. A method of preventing, reducing, and / or ameliorating fatigue symptoms of Intestinal Bowel Syndrome or D-IBS in a subject in need thereof, the method comprising administering Bifidobacterium breve or Bifidobacterium breve strain deposited as DSM 32356 to said subject in need thereof.

12. A method of preventing, reducing, and / or ameliorating fatigue symptoms of Intestinal Bowel Syndrome or D-IBS in a subject in need thereof according to claim 11, wherein Bifidobacterium breve or Bifidobacterium breve strain deposited as DSM 32356 is administered in an amount according to claims 4 to 6.

13. A method of preventing, reducing, and / or ameliorating fatigue symptoms of Intestinal Bowel Syndrome or D-IBS in a subject in need thereof according to claim 11 or 12, wherein Bifidobacterium breve or Bifidobacterium breve strain deposited as DSM 32356 is administered in a composition according to claims 7 to 9.

14. A method of preventing, reducing, and / or ameliorating fatigue symptoms of Intestinal Bowel Syndrome or D-IBS in a subject in need thereof according to claims 11 to 13, wherein Bifidobacterium breve or Bifidobacterium breve strain deposited as DSM 32356 is administered as a capsule according to claim 10.

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