Compositions and methods for using at least one strain of Staphylococcus carnosus for treatment or prevention.

Abstract

The composition contains at least one Staphylococcus carnosus strain, e.g., at least one of S. carnosus CNCM I-5398 or S. carnosus CNCM I-5400. A unit dosage form of the composition contains a prophylactically or therapeutically effective amount of at least one Staphylococcus carnosus strain. A method of making such a composition includes adding at least one Staphylococcus carnosus strain to at least one other food ingredient. Methods of using such compositions for treatment or prevention include promoting mucosal healing; controlling gut microbiota dysbiosis; and treating or preventing intestinal inflammatory diseases such as IBD, irritable bowel syndrome, hepatitis (NASH, NAFLD, alcohol-induced liver injury), allergies, atopy, bone inflammation, rheumatoid arthritis, systemic lupus, Gougerot-Sjogren's syndrome, Reiter's syndrome, poliomyelitis, dermatomyositis, thyroiditis, Graves', Hashimoto's disease, type I diabetes, Addison's disease, autoimmune hepatitis, celiac disease, Beermer's disease, multiple sclerosis, myasthenia gravis, ophthalmia, obesity-related inflammation, age-related low-grade inflammation, Blau syndrome, Alzheimer's disease, cardiovascular disease, atherosclerosis, metabolic syndrome, type II diabetes, gingivitis, periodontitis, and food sensitivities.

Description

Technical Field

[0001] The present invention relates to compositions and methods for using at least one strain of Staphylococcus carnosus for treatment or prevention.

Background Art

[0002]

[0001] This disclosure generally relates to compositions comprising at least one strain of Staphylococcus carnosus, such as at least one of S. carnosus CNCM I-5398 (NCC 971) or S. carnosus CNCM I-5400 (NCC 1052), methods of making such compositions, and methods of using such compositions for treatment or prevention.

[0003]

[0002] Chronic inflammation is at the core of many human diseases, including inflammatory bowel disease (IBD), liver disease, and associated metabolic diseases, and multiple sclerosis. Most of the anti-inflammatory therapies currently in use, such as corticosteroids and antibodies that target specific immunological pathways, have harmful side effects that limit their long-term usefulness.

[0004]

[0003] Furthermore, in this regard, IBD is a group of inflammatory conditions of the colon and small intestine. This disease can cause severe abdominal pain and nutritional problems (food intolerances and deficiencies). The main types of IBD are Crohn's disease (CD) and ulcerative colitis (UC). CD and UC differ mainly by the location and nature of these inflammatory changes. CD can affect any part of the gastrointestinal tract from the mouth to the anus, and the more common clinical symptoms occur in the ileum and large intestine. UC is limited to the colon and rectum.

[0005]

[0004] The etiology of IBD is not yet fully understood. IBD is characterized not only by mucosal inflammation but also by severe damage to the intestinal barrier function. Recent clinical studies have characterized "mucosal healing" as the most significant prognostic factor for long-term remission in IBD patients and for low-risk surgical intervention in CD patients.

[0006]

[0005] Clinical mucosal healing is defined as the complete repair of epithelial and underlying tissues at both the endoscopic and microscopic levels. Mucosal healing reduces the risk of recurrence in patients with inflammatory bowel disease, but the role of dietary supplementation in this process has not been sufficiently studied. [Overview of the project]

[0007]

[0006] In the preliminary context of this disclosure, it should be noted that tryptophan is an essential amino acid for humans and is supplied by dietary proteins. Most of this amino acid is absorbed in the small intestine and metabolized via the kynurenine and 5-hydroxyindole pathways, and unabsorbed tryptophan is catabolized in the small and colon by commensal bacteria. Examples of catabolic products include indole, tryptamine, indole-ethanol, indole-propionic acid, indole-lactic acid (ILA), indole-acetic acid (IAA), skatole, indole-aldehyde (IAld), and indole-acrylic acid.

[0008]

[0007] These metabolites can influence mucosal homeostasis, appetite control, gastrointestinal motility, and / or immune responses mediated by intestinal epithelial cells. In particular, several tryptophan metabolites, such as IAA, IAld, IA, ILA, tryptamine, and skatole, act on aryl hydrocarbon receptors (AHRs) found in intestinal immune cells, thereby modulating immune responses in an AHR-dependent manner. In addition, they act via G protein receptors and signaling pathways, including nuclear factor erythroid 2-related factor 2 (Nrf2), to regulate oxidative stress and inflammatory responses.

[0009]

[0008] The inventors, without being bound by theory, believe that S. carnosus can potentially produce tryptophan metabolites or indole derivatives via catabolic enzymes due to their mucosal homeostatic and immunomodulatory effects, and therefore can improve the gut health of IBD patients or the general population. Tryptophan intake, as an essential amino acid, affects the physiological levels of tryptophan in the body (i.e., blood, brain, and gut), and consequently stimulates serotonin / melatonin synthesis via the aforementioned pathways. Initial levels of tryptophan in food can affect not only the bioavailability of tryptophan but also the availability of tryptophan metabolites in the gut. Therefore, aspects of the present disclosure are based on regulating (e.g., increasing) tryptophan metabolites produced by the gut microbiome.

[0010]

[0009] In this regard, food-grade and / or food-derived microbial strains have a long history of human exposure with a good safety profile, and some of these strains have been developed as probiotics to alleviate certain conditions such as gastrointestinal disorders, e.g., constipation, diarrhea, pain, and / or abdominal bloating. The mechanisms by which probiotics provide relief are not fully understood, and therefore, the selection of specific strains has been largely an empirical challenge. Nevertheless, probiotics are generally considered safe, have a history of safe consumption, and are used in food production.

[0011]

[0010] More recently, as the interaction between the gut microbiome and the host has become better understood, it is becoming clear that the benefits that bacteria provide to the host are often mediated by specific molecules produced by the bacteria. With this in mind, we investigated host physiological pathways in which bacterial metabolites may be involved and selected the aryl hydrocarbon receptor (AHR) pathway as a target.

[0012]

[0011] AHR acts as an environmental sensor by recognizing a family of molecules called indole derivatives, which include metabolites produced by the breakdown of tryptophan. Both the host and certain bacteria can catabolize tryptophan into relevant molecules that are involved in AHR and stimulate many downstream effector functions, which in the gut include the proliferation of regulatory T cells (Tregs) and epithelial cells, among other activities. The net effect of Treg and epithelial cell proliferation is the suppression of inflammation and the promotion of mucosal healing / restoration of mucogutary intestinal function. Patients with IBD have an abnormal complementation (dysbiosis) of the gut microbiota, and as a result, their gut microbiota has a reduced ability to catabolize tryptophan and activate AHR. Several other inflammatory diseases are likely to be associated with a reduced ability of the gut microbiota to catabolize tryptophan. Furthermore, antibiotic treatment is known to negatively affect the healthy balance of gut bacteria, which can lead to impaired physiological and physical (e.g., barrier) functions, as well as reduced defense against pathogenic organisms.

[0013]

[0012] This disclosure addresses the reduced ability of the altered gut microbiota to produce beneficial metabolites and maintain protective functions by processing tryptophan into bioactive metabolites involved in AHR and supplying the host with safe, food-grade and / or food-derived bacteria that have a high capacity to activate multiple health-promoting functions.

[0014]

[0013] Most interventions to address chronic inflammation have adverse effects on the host, but food bacteria have a long history of safe consumption by humans and are suitable for long-term use. Therefore, the use of beneficial bacteria to supplement the important physiological capabilities associated with a healthy gut microbiota in dysbiotic hosts may be a promising approach.

[0015]

[0014] Further features and advantages are described herein and will become apparent from the following drawings and embodiments for carrying out the invention. [Brief explanation of the drawing]

[0016] [Figure 1] This outlines the best NCC species. [Figure 2] This is a table of selected S. carnosus strains of NCC in the first experimental example disclosed herein. [Figure 3] The evaluation of S. carnosus in a mouse DSS colitis model in a second experimental example disclosed herein is shown. [Figure 4] This heatmap shows the changes in cytokine levels in colon tissue collected at the end of the experiment (day 14). The values ​​for the test group are shown in comparison to the DSS vehicle control (shown as "0"). [Figure 5] This chart shows the disease activity index (DAI) at the peak of the disease (day 10) in the second experimental example disclosed herein. A statistical comparison with the vehicle control is shown. [Figure 6A] This chart shows that a bacterial tryptophan metabolite activates AHR in a dose-dependent manner in a third experimental example disclosed herein. [Figure 6B] This chart shows that a bacterial tryptophan metabolite activates AHR in a dose-dependent manner in a third experimental example disclosed herein. [Figure 7A] In a third experimental example disclosed herein, a chart is presented demonstrating that bacterial tryptophan metabolites exhibit low levels of activation and limited enhancement of AHR compared to TCDD. [Figure 7B] In a third experimental example disclosed herein, a chart is presented demonstrating that bacterial tryptophan metabolites exhibit low levels of activation and limited enhancement of AHR compared to TCDD. [Figure 8] This chart shows the results from the initial testing of metabolites in the culture supernatant in the third experimental example disclosed herein. All analytes were quantified from a single sample. [Figure 9] Showing the NST-04 mouse colon; mean total colitis score. Group mean + / − standard error of the mean (SEM). In mice administered DSS, in NCC971- or anti-p40-treated mice, the total colitis score in the distal part was decreased compared to the vehicle control treatment. The same decrease was not observed in the proximal colon. In mice treated with NCC1052 or the combination of NCC971 + NCC1052, the comparative decrease in the proximal and distal parts of the colon was minimal or non-existent. [Figure 10] Showing the proximal colon of NST-04 mice; mean histopathology score. Group mean + / − SEM. The trend among groups was similar to the trend observed in the total colitis score in the proximal part. [Figure 11] Showing the distal colon of mice; mean histopathology score. Group mean + / − SEM. The trend among groups was similar to the trend observed in the total colitis score in the distal part. [Figure 12] Showing the proximal colon of NST-04 mice; mean goblet cell abundance score. Group mean + / − SEM. The abundance of goblet cells mainly increased. In relation to anti-p40 treatment, the decrease and increase in the abundance score were slightly decreased compared to the vehicle control, but this did not lead to a difference in the total score. [[ID=​​​Shows the NST-04 mouse colon; measurement of the average sub-cryptal mucosal thickness. Group mean + / - SEM. In mice administered DSS, in mice treated with NCC971 or anti-p40, the thickness measurements in the distal colon were decreased compared to vehicle control treatment. These same decreases were not observed in the proximal colon. In mice treated with NCC1052 or the combination of NCC971 + NCC1052, no comparative decreases were observed in the proximal and distal parts of the colon.

Mode for Carrying Out the Invention

[0017]

[0029] Definitions

[0030] Several definitions are shown below. However, there may be cases where the definitions are in the section "Embodiments" below, and the above heading "Definitions" does not mean that such disclosure in the section "Embodiments" is not a definition.

[0018]

[0031] All percentages described in this specification are by the total weight of the composition, unless otherwise specified. As used herein, "about", "approximately", and "substantially" refer to numbers within a certain range of a numerical value, for example, within a range of -10% to +10% of the reference number, preferably within a range of -5% to +5% of the reference number, more preferably within a range of -1% to +1% of the reference number, and most preferably within a range of -0.1% to +0.1% of the reference number. All numerical ranges in this specification should be understood to include all integers or fractions within that range. Further, these numerical ranges should be construed to support claims directed to any number or subset of numbers within this range. For example, the disclosure of 1 to 10 should be construed to support ranges such as 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

[0019]

[0032] When used in this disclosure and the attached claims, the singular forms "a," "an," and "the" include multiple references unless otherwise indicated. Thus, for example, a reference to "a bacterial strain" or "the bacterial strain" includes two or more bacterial strains.

[0020]

[0033] The terms “comprise,” “comprises,” and “comprising” should be interpreted as not being exclusive but potentially encompassing others. Similarly, the terms “include,” “including,” and “or” should all be interpreted as potentially encompassing others unless such interpretation is clearly prevented by the context. However, compositions disclosed herein may not include elements not specifically disclosed herein. Therefore, disclosures of embodiments using the term “comprising” include disclosures of embodiments that “consist essentially of” the specified components, and disclosures of embodiments that “consist of” the specified components.

[0021]

[0034] The terms "at least one of X or Y" and "and / or Y" as used in their respective contexts should be interpreted as "X" or "Y" or "X and Y". For example, "at least one of S. carnosus CNCM I-5398 or S. carnosus CNCM I-5400" should be interpreted as "S. carnosus CNCM I-5398 without S. carnosus CNCM I-5400", or "S. carnosus CNCM I-5400 without S. carnosus CNCM I-5398", or "both S. carnosus CNCM I-5398 and S. carnosus CNCM I-5400". These strains may be used individually, in combination with each other, and / or in combination with other strains of S. carnosus.

[0022]

[0035] The term "at least one" should be interpreted as including one or more, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. The term "at least one Staphylococcus carnosus strain" should be interpreted as including a single Staphylococcus carnosus strain, as well as combinations of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more strains. In the context of “at least one” Staphylococcus carnosus strain producing one or more of tryptamine, indole, indolepropionic acid, 3-methylindole, indole-3-acetic acid, or indole-3-acetaldehyde, the term should be interpreted as including one or more strains of Staphylococcus carnosus, e.g., strains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, where each strain produces one or more of tryptamine, indole, indolepropionic acid, 3-methylindole, indole-3-acetic acid, or indole-3-acetaldehyde. Each strain of Staphylococcus carnosus may produce one or more of tryptamine, indole, indolepropionic acid, 3-methylindole, indole-3-acetic acid, or indole-3-acetaldehyde. When two or more strains exist in combination, each strain may produce the same or different one or more metabolites. For example, “at least one Staphylococcus carnosus strain producing one or more of tryptamine, indole, indolepropionic acid, 3-methylindole, indole-3-acetic acid, or indole-3-acetaldehyde” includes any combination of strains producing any combination of the listed metabolites, where each strain may produce only one metabolite or any combination of multiple metabolites.

[0023]

[0036] As used herein, the terms “example” and “such as” are merely illustrative and descriptive, and should not be considered exclusive or comprehensive, especially when followed by a list of terms. As used herein, “associated with” or “linked with” another state means that these states occur simultaneously, preferably that they are caused by the same underlying condition, and most preferably that one of the specified states is caused by the other specified state.

[0024]

[0037] "Prevention" includes reducing the risk and / or severity of a condition or disease. The terms "treatment / therapy" and "to treat / medicate" include both preventive or deterrent treatments (treatments that prevent and / or delay the onset of a diseased condition or disorder in question) and curative, therapeutic, or disease-modifying treatments, such as therapeutic measures for the cure, delay, reduction of symptoms, and / or cessation of progression of a diagnosed diseased condition or disorder, as well as treatment of patients at risk of developing a disease, suspected of having a disease, and patients who are unwell or diagnosed with a disease or medical condition. The terms "treatment / therapy" and "to treat / medicate" do not necessarily mean treating until the subject is fully recovered. The terms "treatment / therapy" and "to treat / medicate" also refer to maintaining and / or promoting the health of an individual who is not currently suffering from a disease but is susceptible to unhealthy conditions. The terms “treatment / therapy” and “to treat / therapy” are also intended to include the synergistic effect, or otherwise enhancement, of one or more primary preventive or therapeutic means. In non-limiting examples, treatment / therapy may be performed by a patient, caregiver, physician, nurse, or other healthcare professional.

[0025]

[0038] As used herein, a preventive or therapeutic “effective dose” is an amount that, in an individual, prevents a deficiency, treats a disease or medical condition, or, more generally, reduces symptoms, alters the expression of a biomarker associated with a disease or condition, manages the progression of a disease, or provides a nutritional, physiological, or medical benefit to the individual. Relative terms such as “promote,” “improve,” “increase,” and “enhance” indicate the level of a property of a subject (e.g., intestinal mucosal function, goblet cell function, or any other indicator of intestinal health) after administration of a composition disclosed herein containing at least one S. carnosus strain, relative to the level of the property immediately prior to administration.

[0026]

[0039] As used herein, the terms “food,” “food product,” and “food composition” mean a product or composition intended for oral ingestion by humans or other mammals and containing at least one nutrient for humans or other mammals.

[0027]

[0040] When used herein, “nutritional composition” and “nutritional product” include any number of food ingredients and optionally optional additional ingredients, based on the functional needs of the product and in full compliance with all applicable regulations. Optional ingredients may include, but are not limited to, conventional food additives such as one or more acidulants, additional thickeners, pH-adjusting buffers or pH adjusters, chelating agents, colorants, emulsifiers, excipients, flavorings, minerals, penetrants, pharmaceutically acceptable carriers, preservatives, stabilizers, sugars, sweeteners, flavorings, and / or vitamins. Optional ingredients may be added in any appropriate amount.

[0028]

[0041] "Probiotics" refers to preparations or components of microbial cells that have beneficial effects on the health or well-being of the host. (Salminen S, Ouwehand A. Benno Y. et al., "Probiotics: how should they be defined," Trends Food Sci. Technol., 1999:10 107-10).

[0029]

[0042] As used herein, the term “unit dosage form” refers to a physically divided unit suitable as a dosage unit for human and animal subjects, each unit containing a predetermined amount of the composition disclosed herein, in an amount sufficient to produce the desired effect, along with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications of a unit dosage form are determined by the specific compounds used, the effect to be achieved, and the pharmacodynamics relating to each compound in the host body.

[0030]

[0043] The "subject" or "individual" is a mammal, preferably a human.

[0031]

[0044] The shares disclosed in this application are deposited with the depositary institutions shown in the table below (Table 1), and are assigned the following deposit dates and accession numbers: [Table 1]

[0032]

[0045] CNCM refers to the Collection nationale de cultures de micro-organismes (Pasteur Institute, 28, rue du Dr Roux, F-75724 Paris Cedex 15, France).

[0033]

[0046] Shares 1 through 5 are held in deposit by Nestec SA (avenue Nestle 55, 1800 Vevey, Switzerland). Subsequently, Nestec SA merged with Societe des Produits Nestle SA, and therefore, according to Article 2(ix) of the Budapest Convention, Societe des Produits Nestle SA is the successor to the rights of Nestec SA.

[0034]

[0047] Embodiment

[0048] As detailed in the experimental examples described herein, in silico screening of a food-grade bacterial library for genes encoding tryptophan catabolic enzymes identified Staphylococcus carnosus strains possessing multiple genes for enzymes that are part of an established tryptophan catabolism pathway. These strains are expected to produce multiple tryptophan catabolic products based on the presence of multiple genes. These degradation molecules are bioactive molecules that can bind to mammalian aryl hydrocarbon receptors (AHRs) and trigger a cascade of downstream effector functions.

[0035]

[0049] Furthermore, targeted metabolomics screening of bacterial culture supernatant confirmed that the metabolites predicted by in silico screening were indeed produced during culture. In addition, evaluation of the purified metabolites in in vitro mouse and human AHR binding and activation assays demonstrated that the metabolites bound to mouse and human receptors and activated the reporter gene. Activation levels were found to be higher in the human AHR receptor compared to the mouse. Importantly, the levels and enhancement of activation were approximately 2,000 times lower than those observed with the host-toxic reference molecule dioxin.

[0036]

[0050] Furthermore, evaluation of various S. carnosus strains in a mouse colitis model identified specific strains that could significantly reduce disease symptoms. Additionally, evaluation of colon tissue cytokines in mouse colitis studies demonstrated an overall suppression of pro-inflammatory cytokines in mice administered with the effective strains.

[0037]

[0051] The identified Staphylococcus carnosus strain likely has a higher number of tryptophan catabolic enzymes and a greater ability to produce these bioactive metabolites compared to other probiotic strains, including Lactobacillus strains such as L. hilgardii, L. farraginis, L. buchneri, L. fermentum, L. reuteri, and L. paracasei.

[0038]

[0052] Accordingly, aspects of the present disclosure are methods for treating osmidrosis in an individual having inflammation, comprising the step of administering to the individual a therapeutically effective dose of at least one Staphylococcus carnosus strain producing one or more of tryptamine, indole, indolepropionic acid, 3-methylindole, indole-3-acetic acid, or indole-3-acetaldehyde. For example, the at least one Staphylococcus carnosus strain includes, individually or in combination with, the strains listed in Table 1, for example, at least one of S. carnosus CNCM I-5398 or S. carnosus CNCM I-5400. In particular aspects, the S. carnosus strain is S. carnosus CNCM I-5398 or S. carnosus CNCM I-5400 individually or in combination thereof. At least one effective amount of Staphylococcus carnosus strain can catabolize tryptophan into metabolites that bind to the organism's aryl hydrocarbon receptor (AHR) and stimulate epithelial cell proliferation.

[0039]

[0053] Further embodiments of the present disclosure relate to a method for treating, preventing, reducing the incidence and / or severity of inflammation in inflammatory bowel diseases, such as IBD, by administering a therapeutically effective or prophylactically effective dose of at least one Staphylococcus carnosus strain producing one or more of tryptamine, indole, indolepropionic acid, 3-methylindole, indole-3-acetic acid, or indole-3-acetaldehyde, for example, at least one of the Staphylococcus carnosus strains listed in Table 1, for example, S. carnosus CNCM I-5398 or S. carnosus CNCM I-5400. In certain embodiments, the S. carnosus strain may be S. carnosus CNCM I-5398 alone or S. carnosus CNCM I-5400 alone or in combination thereof. At least one effective amount of Staphylococcus carnosus strain can catabolize tryptophan into metabolites that bind to the organism's aryl hydrocarbon receptor (AHR) and stimulate the proliferation of regulatory T cell (Treg) epithelial cells.

[0040]

[0054] Another aspect of the present disclosure is a method for promoting intestinal mucosal healing by administering a therapeutic or prophylactic effective dose of at least one Staphylococcus carnosus strain producing one or more of tryptamine, indole, indolepropionic acid, 3-methylindole, indole-3-acetic acid, or indole-3-acetaldehyde, for example, at least one of the Staphylococcus carnosus strains listed in Table 1, e.g., S. carnosus CNCM I-5398 or S. carnosus CNCM I-5400. In certain aspects, the S. carnosus strain may be S. carnosus CNCM I-5398 alone or S. carnosus CNCM I-5400, or a combination thereof. An effective dose of at least one Staphylococcus carnosus strain can catabolize tryptophan into metabolites that bind to the individual's aryl hydrocarbon receptor (AHR) and stimulate epithelial cell proliferation.

[0041]

[0055] A further aspect of this disclosure is a method for controlling or treating or preventing a disorder associated with intestinal microbiota dysbiosis by administering a therapeutically effective or prophylactic dose of at least one Staphylococcus carnosus strain producing one or more of tryptamine, indole, indolepropionic acid, 3-methylindole, 3-methylindole, indole-3-acetic acid, or indole-3-acetaldehyde, for example, at least one of the Staphylococcus carnosus strains listed in Table 1, for example, S. carnosus CNCM I-5398 or S. carnosus CNCM I-5400. In a particular aspect, the S. carnosus strain is S. carnosus CNCM I-5398 or S. carnosus CNCM I-5400.

[0042]

[0056] In any embodiment of the present invention, the strains of Staphylococcus carnosus listed in Table 1 may be used alone or in any combination with other strains of Staphylococcus carnosus listed in Table 1, and / or other Staphylococcus carnosus strains not listed in Table 1 that produce one or more of tryptamine, indole, indolepropionic acid, 3-methylindole, indole-3-acetic acid, or indole-3-acetaldehyde. For example, in any aspect of the present invention, at least one strain of S. carnosus may be S. carnosus CNCM I-5398 alone or S. carnosus CNCM I-5400 in combination. The present invention also provides an edible composition or food composition comprising a therapeutic or prophylactic amount of at least one Staphylococcus carnosus strain producing one or more of tryptamine, indole, indolepropionic acid, 3-methylindole, 3-methylindole, indole-3-acetic acid, or indole-3-acetaldehyde, for example, at least one of the Staphylococcus carnosus strains listed in Table 1, for example, S. carnosus CNCM I-5398 or S. carnosus CNCM I-5400. In certain embodiments, the S. carnosus strain is S. carnosus CNCM I-5398 alone or in combination with S. carnosus CNCM I-5400. Edible compositions or food compositions can be selected from the group consisting of complete nutrition products, beverages, dietary supplements, meal replacements, food additives, supplements for food products, powders for dissolving, enteral nutrition products, infant formulas, and combinations thereof.

[0043]

[0057] In one embodiment, the present invention also provides an edible composition or food composition comprising a therapeutic or prophylactic effective amount of at least one Staphylococcus carnosus strain producing one or more of tryptamine, indole, indolepropionic acid, 3-methylindole, 3-methylindole, indole-3-acetic acid, or indole-3-acetaldehyde for use in the treatment of inflammation in an individual. The at least one Staphylococcus carnosus strain may be at least one of the strains listed in Table 1, for example, S. carnosus CNCM I-5398 or S. carnosus CNCM I-5400. In a particular embodiment, the S. carnosus strain may be S. carnosus CNCM I-5398 alone or in combination with S. carnosus CNCM I-5400.

[0044]

[0058] At least one Staphylococcus carnosus strain has 1 x 10 3 ~1 × 10 12 Preferably 1 × 10 7 ~1 × 10 11 It can be administered to an individual as a daily dose of cfu (cfu = colony-forming unit). At least one Staphylococcus carnosus strain contains 1 × 10⁻⁶ 3 ~1 × 10 12 It may be administered in a composition containing a dry composition of cfu / g.

[0045]

[0059] At least one Staphylococcus carnosus strain may be alive, fragmented, or in the form of a fermentation product (e.g., supernatant) or metabolite, or any mixture thereof.

[0046]

[0060] In some embodiments, at least one Staphylococcus carnosus strain is administered in a composition further comprising one or more additional probiotics, where at least one Staphylococcus carnosus constitutes the majority of the total amount of probiotics in the composition. For example, the amount of any Lactobacillus in the composition is preferably less than the amount of at least one Staphylococcus carnosus. In some embodiments, at least one Staphylococcus carnosus is the only probiotic in the composition.

[0047]

[0061] At least one Staphylococcus carnosus strain may be administered to an individual by at least one route selected from the group consisting of oral, topical, enteral, and parenteral administration. For example, at least one Staphylococcus carnosus strain may be administered in compositions selected from the group consisting of complete nutritional products, beverages, dietary supplements, meal replacements, food additives, supplements for food products, dissolvable powders, enteral nutrition products, infant formulas, and combinations thereof.

[0048]

[0062] In some embodiments, at least one Staphylococcus carnosus strain can be administered in a dose effective for treating, preventing, reducing the incidence of, and / or reducing the severity of inflammation. Non-limited examples of such inflammation include acute inflammation, skin inflammation, inflammatory bowel disease (IBD), including Crohn's disease and / or ulcerative colitis, irritable bowel syndrome, hepatitis (NASH, NAFLD, alcohol-induced liver injury), allergies, atopic dermatitis, bone inflammation, rheumatoid arthritis, systemic lupus, Gougeleau-Sjögren's syndrome, Reiter's syndrome, poliomyelitis, dermatomyositis, thyroiditis, Graves' disease, Hashimoto's disease, type 1 diabetes, Addison's disease, autoimmune hepatitis, celiac disease, Biermer's disease, multiple sclerosis, myasthenia gravis, ophthalmitis, obesity-associated inflammation, and age-related low-grade inflammation. The group may be selected from the following: inflammation, Blau syndrome, Alzheimer's disease, cardiovascular disease, atherosclerosis, metabolic syndrome, type II diabetes, gingivitis, periodontitis, food sensitivity, celiac disease, and combinations thereof.

[0049]

[0063] Inflammation treated or prevented by at least one Staphylococcus carnosus strain may be IBD, such as Crohn's disease or ulcerative colitis.

[0050]

[0064] In some embodiments, individuals are selected from a group consisting of infants, children, adolescents, adults, and the elderly.

[0051]

[0065] Preferably, at least one Staphylococcus carnosus strain is administered with a composition further comprising at least one component selected from the group consisting of prebiotics, amino acids, proteins, nucleotides, vitamins, fish oil, non-marine omega-3 fatty acid sources, phytonutrients, antioxidants, and mixtures thereof.

[0052]

[0066] Prebiotics are foods that promote the growth of beneficial bacteria in the gut. Prebiotics are not broken down in the stomach of the individual ingesting them, nor are they absorbed into the GI tubule; however, they undergo fermentation by the gastrointestinal microbiota and / or probiotics. The combination of prebiotics with at least one Staphylococcus carnosus strain delivers synergistic health benefits, making the addition of prebiotics beneficial. Compositions containing a combination of prebiotics and probiotics are commonly known as symbiotic compositions.

[0053]

[0067] Prebiotics that can be used with at least one Staphylococcus carnosus strain include, but are not particularly limited, all food substances that promote the growth of probiotics in the intestine. Preferably, prebiotics can be optionally selected from the group consisting of oligosaccharides containing fructose, galactose, and mannose; dietary fiber, especially soluble fiber, soy fiber; inulin; or mixtures thereof. Preferred prebiotics are fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), isomaltoligosaccharides, xylooligosaccharides, soy oligosaccharides, glycosylsucrose (GS), lacto-oligosaccharides (LS), lactulose (LA), palatinose-oligosaccharides (PAO), maltooligosaccharides, pectin, and / or hydrolysates thereof.

[0054]

[0068] A composition containing at least one Staphylococcus carnosus strain may be a food product, an animal food product, or a pharmaceutical composition. For example, the product may be a nutritional composition, a nutraceutical, a beverage, a food additive, or a pharmaceutical.

[0055]

[0069] Food additives or pharmaceuticals may be in the form of, for example, tablets, capsules, lozenges, liquids, or powders in sachets. Food additives or pharmaceuticals are preferably provided as a sustained-release formulation to enable a substantially constant supply over a long period of time of at least one Staphylococcus carnosus strain.

[0056]

[0070] A composition comprising at least one Staphylococcus carnosus strain may preferably be selected from the group consisting of: powdered milk-based products; instant beverages; ready-to-drink formulations; nutritional powders; nutritional liquids; milk-based products, especially yogurt or ice cream; cereal products; beverages; water; coffee; cappuccino; malt beverages; chocolate-flavored beverages; cooked products; soups; tablets; and / or syrups.

[0057]

[0071] The composition may optionally include any milk obtained from animal or plant sources, such as one or more of the following: cow's milk, human milk, sheep's milk, goat's milk, horse's milk, camel's milk, rice milk, or soy milk. Additionally or alternatively, milk protein fractions or colostrum may be used.

[0058]

[0072] A composition comprising at least one Staphylococcus carnosus strain may further contain protective hydrophilic colloids (such as gum, protein, or modified starch), binders, film-forming agents, encapsulating agents / encapsulating materials, wall / shell materials, matrix compounds, coatings, emulsifiers, surfactants, solubilizers (such as oils, fats, waxes, or lecithin), adsorbents, carriers, fillers, co-compounds, dispersants, wetting agents, processing aids (solvents), fluidizers, flavoring agents, bulking agents, gelling agents, gel-forming agents, antioxidants, and antimicrobial agents. A composition containing at least one Staphylococcus carnosus strain may also contain conventional pharmaceutical excipients and adjuvants, excipients and diluents, including but not limited to water, gelatin derived from any raw material, vegetable gum, lignin sulfonate, talc, sugar, starch, gum arabic, vegetable oil, polyalkylene glycol, flavorings, preservatives, stabilizers, emulsifiers, buffers, lubricants, colorants, wetting agents, and fillers. Furthermore, a composition containing at least one Staphylococcus carnosus strain may also contain organic or inorganic carrier materials suitable for oral or enteral administration, as well as vitamins, minerals, trace elements and other micronutrients, in accordance with the recommendations of government agencies such as the USRDA.

[0059]

[0073] A composition comprising at least one Staphylococcus carnosus strain may optionally contain a protein source, a carbohydrate source, and / or a lipid source, particularly in embodiments of the composition that are food products.

[0060]

[0074] Examples include proteins of any suitable food origin, such as animal proteins (milk proteins, meat proteins, and egg proteins, etc.); plant proteins (soybean proteins, wheat proteins, rice proteins, and pea proteins, etc.); mixtures of free amino acids; or combinations thereof. Milk proteins such as casein and whey proteins, and soybean proteins are particularly preferred.

[0061]

[0075] If the composition contains a fat source, the fat source more preferably accounts for 5% to 40% of the formula's energy; for example, 20% to 30% of the energy. DHA may be added. A suitable fat profile can be obtained using a blend of canola oil, corn oil, and high-oleic sunflower oil.

[0062]

[0076] The carbohydrate source can preferably provide 40% to 80% of the energy of the composition. Any suitable carbohydrates, such as sucrose, lactose, glucose, fructose, solid corn syrup, maltodextrin, and mixtures thereof, can be used.

[0063]

[0077] A composition comprising at least one Staphylococcus carnosus strain may be administered to humans or animals, particularly companion animals, pets, or livestock. This composition has beneficial effects on any age group. Preferably, the composition is formulated for administration to infants, young people, adults, or the elderly. In some embodiments, the composition is administered to mothers during pregnancy and lactation to treat infants.

[0064]

[0078] In one embodiment, a composition containing at least one Staphylococcus carnosus strain can be administered for at least 10, 20, 24, 30, 40, 42, 50, or 60 weeks. Preferably, the composition can be administered for 10-60 weeks, 20-50 weeks, 15-30 weeks, or 35-45 weeks.

[0065]

[0079] A composition containing at least one Staphylococcus carnosus strain can maintain or improve the mucosal health of IBD patients, as demonstrated by the maintenance of healthy mucosa as seen by endoscopy.

[0066]

[0080] The maintenance of healthy mucosa as demonstrated by endoscopy can be evaluated by the simple endoscopic score (SES-CD) for Crohn's disease. Therefore, improvement in healthy mucosa as demonstrated by endoscopy can be indicated by a decrease in the mean SES-CD score from the start of administration of a composition containing at least one Staphylococcus carnosus strain to 20-45 weeks, preferably 20-30 weeks, for example, 24 weeks, after the start of administration of a composition containing at least one Staphylococcus carnosus strain (endoscopic improvement).

[0067]

[0081] The maintenance of healthy mucosa as demonstrated by endoscopy can be indicated by the maintenance of an endoscopic response, where the endoscopic response is indicated by a reduction of at least 3 points in SES-CD from the start of administration of the composition containing at least one Staphylococcus carnosus strain for 20 to 45 weeks, preferably 20 to 30 weeks, for example up to 24 weeks after the start of administration of the composition containing at least one Staphylococcus carnosus strain.

[0068]

[0082] The maintenance of healthy mucosa as demonstrated by endoscopy can also be shown by the maintenance of clinical remission, where clinical remission is shown as resulting in a CDAI score of less than 150 at 20 to 45 weeks, preferably 20 to 30 weeks, for example at 24 weeks, after the initiation of administration of a composition containing at least one Staphylococcus carnosus strain.

[0069]

[0083] A composition containing at least one Staphylococcus carnosus strain can extend the time to endoscopic or clinical recurrence. This allows the composition to reduce the economic impact of CD, as indicated by surgery, hospitalization, and CD complication rates.

[0070]

[0084] A composition containing at least one Staphylococcus carnosus strain can improve quality of life, for example, as indicated by IBDQ, SF-36v2, and EQ-5, which are determined 20 to 45 weeks, preferably 20 to 30 weeks, for example 24 weeks, after the start of administration of the composition containing at least one Staphylococcus carnosus strain.

[0071]

[0085] A composition containing at least one Staphylococcus carnosus strain can improve the composition and functionality of the gut microbiome.

[0072]

[0086] A composition comprising at least one Staphylococcus carnosus strain can improve non-invasive biomarkers such as CRP or fecal calprotectin.

[0073]

[0087] Compositions containing at least one Staphylococcus carnosus strain can be combined with common standard treatments for IBD. These standard treatments include surgery, antibiotics, immunosuppressants, and anti-inflammatory drugs. Immunosuppressants can be selected from the group consisting of prednisone, TNF or TNFα inhibitors (e.g., infliximab, adalimumab), azathioprine (Imuran), methotrexate, and 6-mercaptopurine. Preferred anti-inflammatory drugs are mesalamine (USAN) or 5-aminosalicylic acid (5-amino-2-hydroxybenzoic acid, 5-ASA).

[0074]

[0088] Preferably, a composition comprising at least one Staphylococcus carnosus strain is administered in combination with at least one TNF inhibitor or TNF inhibitor therapy. Preferably, at least one TNF inhibitor is a TNF alpha inhibitor. Preferably, at least one TNF alpha inhibitor is infliximab or adalimumab, and most preferably the TNF alpha inhibitor is a combination of infliximab and adalimumab.

[0075]

[0089] Administration of either the above-mentioned immunosuppressant or anti-inflammatory agent in combination with a composition containing at least one Staphylococcus carnosus strain is preferred. This combination results in a synergistic effect of the administered compounds.

[0076]

[0090] A composition containing at least one Staphylococcus carnosus strain can be used to treat IBD, treat subjects in remission of IBD, or prevent or delay relapses of IBD in subjects, wherein the composition containing at least one Staphylococcus carnosus strain is administered in combination with a drug effective against IBD. The drug is preferably an immunosuppressant or 5-aminosalicylic acid (5-ASA). The subject may be a subject who has undergone surgery or is scheduled to undergo surgery. Administration of the composition containing at least one Staphylococcus carnosus strain can be done before, during, or after administration of the drug.

[0077]

[0091] In some embodiments, a composition comprising at least one Staphylococcus carnosus strain can promote intestinal mucosal healing. In such embodiments, a composition comprising at least one Staphylococcus carnosus strain can be administered to an individual with intestinal mucosal damage.

[0078]

[0092] In some embodiments, compositions comprising at least one Staphylococcus carnosus strain can control or treat or prevent disorders associated with gut microbiota dysbiosis. Gut microbiota dysbiosis is a significant deviation from a balanced microbiota in terms of the overall microbiota profile, metabolism, or levels of specific taxa. Gut microbiota dysbiosis is typically associated with disease and increases vulnerability to disease. For example, a decrease in levels of the Bifidobacterium genus is associated with an increased risk of infection and other medical conditions in infants.

[0079]

[0093] Microbiota dysbiosis can be triggered, for example, by cesarean section, premature birth, intrauterine exposure to antibiotics, during or after childbirth, parenteral feeding, hospitalization, or psychological stress. Microbiota dysbiosis can also lead to gastrointestinal dysfunction (indigestion, motility disorders, gastroesophageal reflux disease, slow transit, oral feeding intolerance, constipation, diarrhea), Hirschsprung's disease, short bowel syndrome, gastrointestinal infections, and inflammation affecting the gastrointestinal tract (such as necrotizing enterocolitis), as well as obstructive lesions.

[0080]

[0094] These conditions are not only a consequence of gastrointestinal disorders, but microbiome dysbiosis can actually cause them. Thus, microbiome dysbiosis can lead to, for example, digestive disorders, motility disorders, gastroesophageal reflux disease, slow gastrointestinal transit, oral feeding intolerance, Hirschsprung's disease, as well as inflammation affecting the gastrointestinal tract (such as necrotizing enterocolitis) and obstructive lesions.

[0081]

[0095] A composition comprising at least one Staphylococcus carnosus strain can prevent or treat microbiome dysbiosis in mammals at risk of or suffering from microbiome dysbiosis, or can prevent or treat disorders associated with microbiome dysbiosis.

[0082]

[0096] Disorders that can be treated or prevented by controlling microbiome dysbiosis include, for example, infectious tendencies, allergies, type 1 diabetes, insulin resistance, type 2 diabetes, celiac disease, peripheral and central obesity, obesity, inflammatory bowel diseases such as necrotizing enterocolitis, Crohn's disease, and ulcerative colitis, as well as functional gastrointestinal disorders such as functional diarrhea, functional constipation, recurrent umbilical colic, and dyspepsia.

[0097] [Examples]

[0083]

[0098] The following non-limiting examples are generally illustrative of embodiments of the present disclosure. In this regard, the inventors selected strains based on genomes capable of producing multiple tryptophan metabolites. These strains effectively produce their metabolites (in vitro culture); the metabolites produced are effective AHR receptor agonists (and to an effective balance); and when tested in animal DSS models, the strains are indeed effective and reduce disease scores, as well as affecting only pro-inflammatory cytokine production and anti-inflammatory cytokine production.

[0084]

[0099] Example 1

[0100] The objective was to identify microbial strains possessing the enzymes necessary to produce tryptophan-derived molecules that enable AHR activation and the generation of samples for metabolomics evaluation, using in silico methods. Details are provided below. Microbial strains possessing the necessary genes encoding enzymes that produce tryptophan-derived metabolites such as tryptamine, indole, indole-3-acetaldehyde, and indole-3-acetic acid were screened in silico. Strains belonging to the traditional meat starter culture, Staphylococcus carnosus, were identified as promising candidates, and samples were prepared for further metabolomics evaluation.

[0085]

[0101] Methods and Results

[0102] We searched the publicly available Uniprot database for publicly available protein references of enzymes that catalyze the formation of tryptamine, indole, indole-3-acetaldehyde, indole-3-acetic acid, and 3-methyl-indole from tryptophan (Table 1). Reference proteins for indole acetate oxidase were not available in the publicly available databases Uniprot, Swissprot, and BRENDA. [Table 2]

[0086]

[0103] To identify the genes capable of encoding the above enzymes, a sequence similarity search was performed on the entire coding sequence of the Nestle Culture Collection (NCC) genome. The search yielded a total of 1,339 NCC strains that matched at least one of the enzymes. Of these, 49 strains matched three of these enzymes.

[0087]

[0104] Some of the reference proteins used in the search originate from organisms with relatively weak phylogenetic relationships to the NCC strains, so the identity percentage may be relatively low. However, annotation of these genes confirmed the precise potential activity of the enzymes they encode. A summary of the best NCC species is shown in Figure 1.

[0088]

[0105] As shown in Figure 1, Staphylococcus carnosus strain exhibits intriguing diversity in the production of potential metabolites, differing from previously discovered probiotics, primarily belonging to the Lactobacillus species. Staphylococcus carnosus strain is particularly interesting because it is widely used in the food industry as a meat starter, and industrially suitable growth media have already been developed.

[0089]

[0106] From the NCC strains, S. carnosus isolates and fully sequenced strains were selected for further testing. All of these strains were reactivated under optimal conditions. Specifically, growth in Trypticase soy yeast (TSY) broth at 37°C for 10–48 hours at 220 rpm was suitable for all strains. Enterococcus feacalis NCC 1978 was selected as a negative control because it did not contain the necessary genes and grew well in TSY.

[0090]

[0107] The strains and their associated supernatants were cultured in a homogeneous manner for further metabolomics evaluation. Therefore, the growth profiles of each strain were obtained in duplicate. Specifically, each strain was inoculated at 2% from newly grown cultures and incubated at 37°C with stirring at 500 rpm.

[0091]

[0108] To prepare the pellet and supernatant, each S. carnosus strain was cultured separately in 10 mL of TSY broth medium at 37°C and 220 rpm. As previously determined, cells and supernatant were collected 3 hours after cell entry into the stationary phase. Briefly, 10 mL of the culture was centrifuged (3500 rpm, 20 minutes). The supernatant was collected and frozen at -80°C for analysis.

[0092]

[0109] To evaluate the specific amounts of tryptophan metabolites in various matrices, LC-MS methods were developed and validated targeting each metabolite. In summary, NCC strains possessing the necessary genes encoding enzymes that produce tryptophan-derived metabolites such as tryptamine, indole, indole-3-acetaldehyde, and indole-3-acetic acid were screened in silico. Strains belonging to Staphylococcus carnosus, a traditional meat starter culture, were identified as promising candidates, and samples were prepared for further metabolomics evaluation.

[0093]

[0110] Example 2

[0111] We select a probiotic strain of Staphylococcus carnosus as a source of tryptophan metabolites to improve the gut health of IBD patients and / or the general population. The targeted efficacy is AHR activation and reduced inflammatory potential in the gut. The scientific hypothesis was that tryptophan metabolites produced by S. carnosus could lead to activation of aryl hydrocarbon receptors (AHRs) in intestinal epithelial cells to improve / enhance the integrity of these cells.

[0094]

[0112] The target population is either inflammatory bowel disease (IBD) patients or the general population who are scheduled to take S. carnosus daily.

[0095]

[0113] Methods and Results

[0114] The NCC genome database was screened for enzymes that catabolize tryptophan and related substrates. Following in vitro growth and recovery studies, nine strains were selected for further in vivo screening studies. The nine selected strains were NCC836, NCC846, NCC888; NCC971, NCC981, NCC1052, NCC1084, NCC1090, NCC1109, and NCC1978. After animal testing in DSS model mice, two strains (NCC971 and NCC1052) were selected due to their higher recovery rates in weight gain in these animal models compared to the other strains. NCC971 is also known as CNCM I-5398, NCC1053 is also known as CNCM I-5400, NCC846 is also known as CNCM I-5423, NCC982 is also known as CNCM I-5399, and NCC1090 is also known as CNCM I-5401.

[0096]

[0115] In silico screening for pathogenic factors and antibiotic resistance was evaluated internally as follows.

[0097]

[0116] The genes for SE toxins (Staphylococcal enterotoxin (SE), hemolysin, exfoliative toxin A (ETA), and toxic shock syndrome toxin 1) were not identified in strains NCC1052 and NCC971 (CNCMI-5400 and CNCMI-5398, respectively).

[0098]

[0117] Whole genome sequences are available for all S. carnosus NCC strains tested. These additional S. carnosus NCC strains were screened in silico for the presence of the five enzyme sequences found in NCC1052 and NCC971 strains (CNCM I-5400 and CNCM I-5398, respectively) using the same similarity search tool with the same filtering threshold. Most strains (16 / 18) were found to be positive for these five proteins.

[0099]

[0118] In silico screening for antibiotic resistance was performed on 19 S. carnosus NCC strains with whole-genome sequencing, including NCC1052 and NCC971 (CNCM I-5400 and CNCM I-5398, respectively), using a reference database. Screening was performed using "strict" and "loose" predictions. Due to the low confidence level of the "loose" predictions, all of these hits were further filtered while maintaining identity percentages of over 70%. Most of the strains, including NCC1052 and NCC971, were not hit by the "strict" predictions. Using the "loose" predictions resulted in hits of fewer than 10 CARD database proteins associated with potential antibiotic resistance. In silico predictions using the "strict" predictions did not identify any antibiotic resistance in the NCC1052 and NCC971 strains.

[0100]

[0119] To screen for the best candidate to improve gut health from nine S. carnosus strains, we used a mouse IBD (inflammatory bowel disease) model assisted by DSS (dextran sodium sulfate) (Figure 3). Mice were fed via gastric tube for 10 minutes. 9 Each CFU strain was pre-treated daily for 7 days (D-7), followed by treatment with DSS (3%) and the strain for 5 days, and then each strain alone for a further 9 days (D14). Readout included daily weight change, daily composite disease activity index (DAI), endoscopy, and colon weight:length ratio score (Figure 4). DAI is a composite score of weight loss, diarrhea, bloody stool, and activity level.

[0101]

[0120] The combined DAI scores of NCC971 and NCC1052 (CNCM I-5398 and CNCM I-5400, respectively) were significantly lower than those of the DSS control group on day 10 (Figure 5). No apparent specific adverse events or deaths were reported in this study.

[0102]

[0121] Example 3

[0122] Using an in vitro AHR binding assay with reporter gene readout, the relative binding activity of tryptophan-derived metabolites produced by S. carnosus was characterized and compared with known toxic ligands. Both human and mouse AHR assays were utilized. Dioxin (TCDD) and indirubin were used as reference compounds with strong binding and activation profiles. Commercially available metabolites were analyzed in dose-response studies. Bacterial tryptophan metabolites activate AHR in a dose-dependent manner (Figures 6A and 6B).

[0103]

[0123] As shown in Figures 7A and 7B, tryptophan-derived metabolites produced by S. carnosus demonstrate lower levels of AHR activation and limited enhancement compared to TDD (more than 2000 times lower activity than TCDD (dioxin)).

[0104]

[0124] Figure 8 is a chart showing the results of the initial metabolite testing in the culture supernatant.

[0105]

[0125] Example 4

[0126] This example relates to the histopathology of mouse proximal and distal colon samples in a dextran sulfate sodium (DSS)-induced colitis model, and to the evaluation of the efficacy of treatment with NCC971 or NCC1052 (CNCM I-5400 and CNCM I-5398, respectively, either alone or in combination) in reducing lesion severity; for comparison of test substances, vehicle treatment was used as a negative control and anti-p40 treatment was used as a positive control.

[0106]

[0127] Materials and methods

[0128] Mice were administered 3% DSS on days 0-5 and sacrificed on day 19. The treatment was carried out according to the following experimental design; if the number of animals in parentheses differs from the number of animals in the experiment, it indicates the number of animals used for histopathological evaluation.

[0107]

[0129] Experimental Design [Table 3]

[0108]

[0130] Histological method

[0131] After slaughter, colon samples were collected by the sponsor or their designated representative according to the following protocol: the entire colon was rinsed, and the distal 5 cm portion was collected; of this 5 cm portion, the most proximal 2 cm and the most distal 2 cm regions were separated and fixed in 10% neutral buffered formalin. Colon fragments from 96 mice were submitted to Inotiv Boulder. The proximal and distal regions of the collected colon were trimmed into three cross sections per region, and both the proximal and distal regions were embedded in the same block. The block was cut into thin sections of approximately 5 μm thickness and stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS).

[0109]

[0132] Pathological method

[0133] H&E-PAS stained glass slides were evaluated by a committee-certified veterinary pathologist using a light microscope. Colitis lesions (inflammation, glandular necrosis / loss, erosion, thickening, and edema) were assigned a severity score from 0 to 5 (0 = absent / normal, 1 = mild, 2 = mild, 3 = moderate, 4 = severe, 5 = very severe). The total colitis score for each sample was determined by summing the individual histopathological scores (range 0 to 25).

[0110]

[0134] The H&E-PAS slides also included a score for the abundance of goblet cells. This feature was scored based on the presence of residual colonic gland (i.e., areas without colonic gland were excluded; this feature assessed glandular loss (see above)). The abundance of goblet cells was scored compared to untreated samples (score 0); an increase in goblet cells was scored within the positive range, and a decrease in goblet cells was scored within the negative range; a total score was also provided (decrease in goblet cells score + increase in goblet cells score). -3 = Loss of diffuse goblet cell activity -2 = Loss of multifocality in goblet cells -1 = Local loss of goblet cells 0 = Approximately the same amount of goblet cells as in the untreated control sample. 1 = Local increase in goblet cells 2 = Increased multifocal lesions in goblet cells 3 = Diffuse increase in goblet cells

[0111]

[0135] Subcrypt mucosal measurements were performed in three representative regions per section (three sections each for the proximal and distal parts = nine measurements per colonic region; six total cross-sectional areas per animal = 18 measurements per animal). Mucosal measurements (μm) were collected by measuring the distance between the basement membrane of the mucosal crypt and the medial edge of the muscularis mucosa (subcrypt space). Areas completely lacking epithelium (glandular loss / erosion) were not measured. Areas where glandular epithelium had been replaced by stratified squamous epithelium (squamous metaplasia) were avoided whenever possible. In sections where most or all of the epithelium had been replaced by squamous epithelium, measurements were collected between the basement membrane of the squamous basal layer and the medial edge of the muscularis mucosa.

[0112]

[0136] statistical analysis

[0137] Data are presented as mean ± standard error of the mean (SEM). Semi-quantitative severity scores were analyzed using a nonparametric Kruskal-Wallis test with paired Wise-Mann-Whitney direct tests. A two-tailed test was used, and significance was set at p ≤ 0.05 for all tests.

[0113]

[0138] Results and Discussion

[0139] Morphological findings

[0140] Administration of dextran sulfate sodium (DSS) to mice resulted in expected histological lesions. These lesions included subacute inflammation of the mucosa / submucosa, mucosal necrosis / gland loss, erosions, submucosal edema, and epithelial hyperplasia. Subacute inflammation was characterized by infiltration and aggregation of neutrophils, lymphocytes, and macrophages. Mucosal necrosis was characterized by damage, necrosis, or complete loss of colonic glands. Erosions were characterized by necrosis or loss of surface epithelium on the surface of the muscularis mucosa. Submucosal edema was characterized by swelling of the submucosa due to clear space or pale eosinophilic fluid, varyingly accompanied by lymphatic vessel dilation and similar edematous swelling of the lamina propria. Epithelial hyperplasia was characterized by elongation of colonic glands, crypt and glandular branching / dividing, basophilia of epithelial cells, and an increase in the number of epithelial mitotic figures. Some distal colon specimens also exhibited squamous metaplasia, characterized by the replacement of glandular epithelium by laminar squamous epithelium.

[0114]

[0141] This study observed both increases and decreases in goblet cell counts. Generally, decreases in goblet cell counts were seen in glands directly adjacent to or within areas of gland loss and erosion, while increases in goblet cell counts were associated with some areas of hyperplasia.

[0115]

[0142] Most of the "proximal" fragments provided for histopathological evaluation contained either the proximal colon (the colon region with mucosal folds) or / or the midcolon (the region without mucosal folds). Therefore, the results for the proximal colon represent some combination of the proximal and midcolon (see Table 1 for the P:M ratio).

[0116]

[0143] result

[0144] In animals that were not treated with DSS, no colitis lesions were present in either the proximal or distal colon (Group 1).

[0117]

[0145] The total score (Figure 9) was generally higher in distal colon fragments than in proximal colon fragments. Mice treated with NCC971 (group 3; p=0.050) or anti-p40 (group 6; p=0.033) showed a statistically significant reduction in distal colitis total score compared to vehicle controls (group 2). These reductions were not observed in the proximal colon (p>0.2; see discussion). There were no statistically significant reductions associated with NCC1052 (group 4) treatment or the NCC971+NCC1052 (group 5) combination treatment in the proximal (p>0.5) or distal (p>0.3) colon.

[0118]

[0146] Similar intergroup trends were observed in the component histopathology scores of the proximal (Figure 10) and distal (Figure 11) parts of the colon.

[0119]

[0147] DSS-associated goblet cell abundance increased mainly in the proximal colon (Figure 12) and decreased in the distal colon (Figure 13). No statistically significant difference was observed in the total abundance score (p-value > 0.15) in any region of the colon between the vehicle control (group 2) and the treatment groups (groups 3-6).

[0120]

[0148] Subcryptal mucosal measurements (Figure 14) were significantly larger in DSS-treated mice (Groups 2-6) compared to untreated mice (Group 1) in both the proximal (p = 0.009) and distal (p < 0.001) regions. The intergroup trends generally reflected those observed in the total colitis score (Figure 9). Mice treated with NCC971 showed a statistically significant decrease in distal thickness measurements compared to vehicle controls (Group 2) (Group 3; p = 0.011). These decreases were not observed in the proximal colon (p > 0.2). Treatment with the control compound anti-p40 (Group 6) also reduced subcryptal thickness measurements, but the difference was not statistically significant (p = 0.414). In the proximal or distal parts of the colon, there was no reduction associated with the NCC1052 (group 4) treatment or the combination of NCC971 + NCC1052 (group 5) treatment.

[0121]

[0149] Discussion

[0150] DSS administration to mice is known to result in more severe disease severity in the more distal colonic region (i.e., colitis severity: proximal colon < mid-colon < distal colon).3 In this experiment, the proximal colon consisted of true proximal and mid-colon in varying ratios. The ratio of proximal to mid-colon regions (P:M ratio) may influence the overall severity score observed in the proximal region. Typically, a higher P:M ratio (more proximal than mid-colon) is associated with a lower overall severity score.

[0122]

[0151] conclusion

[0152] DSS administration effectively induced the expected histological lesions. The results for the test substance differed between the proximal and distal colon. NCC971 treatment and anti-p40 treatment showed a statistically significant reduction in colitis severity in the distal colon, but not in the proximal colon. NCC1052 treatment, or the combination of NCC971 and NCC1052, generally did not alter the severity of colitis compared to vehicle controls in any colonic region.

[0123]

[0153] It should be understood that various changes and modifications to the current preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of this subject matter and without impairing the intended advantages. Accordingly, such changes and modifications are intended to be covered by the appended claims.

Claims

1. A composition for treating inflammation in an individual having inflammation, comprising a therapeutically effective amount of at least one Staphylococcus carnosus strain, wherein the at least one Staphylococcus carnosus strain produces one or more of the following: tryptamine, indole, indolepropionic acid, 3-methylindole, indole-3-acetic acid, or indole-3-acetaldehyde.

2. A composition for preventing inflammation, reducing the incidence of inflammation, and / or reducing the severity of inflammation in an individual at risk of inflammation, comprising a preventive amount of at least one Staphylococcus carnosus strain, wherein the at least one Staphylococcus carnosus strain produces one or more of the following: tryptamine, indole, indolepropionic acid, 3-methylindole, indole-3-acetic acid, or indole-3-acetaldehyde.

3. A composition for preventing, treating, or controlling microbiome dysbiosis in an individual at risk of or suffering from microbiome dysbiosis, comprising a preventive amount of at least one Staphylococcus carnosus strain, wherein the at least one Staphylococcus carnosus strain produces one or more of the following: tryptamine, indole, indolepropionic acid, 3-methylindole, indole-3-acetic acid, or indole-3-acetaldehyde.

4. A composition for the prevention or treatment of a disorder in an individual at risk of or having said disorder related to microbiome dysbiosis, comprising at least one Staphylococcus carnosus strain producing one or more of tryptamine, indole, indolepropionic acid, 3-methylindole, indole-3-acetic acid, or indole-3-acetaldehyde.

5. A composition for promoting the healing of the intestinal mucosa in an individual requiring healing of the intestinal mucosa, comprising a prophylactic effective amount of at least one Staphylococcus carnosus strain, wherein the at least one Staphylococcus carnosus strain produces one or more of the following: tryptamine, indole, indolepropionic acid, 3-methylindole, indole-3-acetic acid, or indole-3-acetaldehyde.

6. The composition according to any one of claims 1 to 5, wherein the at least one Staphylococcus carnosus strain comprises at least one of S. carnosus CNCM I-5398 or S. carnosus CNCM I-5400.

7. The composition according to any one of claims 1 to 5, wherein the at least one Staphylococcus carnosus strain is administered with a composition further comprising one or more additional probiotics, wherein the amount of any Lactobacillus in the composition is less than the amount of the at least one Staphylococcus carnosus.

8. The composition according to any one of claims 1 to 5, wherein the at least one Staphylococcus carnosus strain is administered in a composition in which the at least one Staphylococcus carnosus is the sole probiotic.

9. The composition according to any one of claims 1 to 5, wherein the at least one Staphylococcus carnosus strain is administered to the individual by at least one route selected from the group consisting of oral, topical, enteral, and parenteral administration.

10. The composition according to any one of claims 1 to 5, wherein the at least one Staphylococcus carnosus strain is administered in a composition selected from the group consisting of complete nutritional products, beverages, dietary supplements, meal replacements, food additives, supplements for food products, dissolvable powders, enteral nutrition products, infant formulas, and combinations thereof.

11. The composition according to claim 1 or 2, wherein the inflammation is selected from the group consisting of acute inflammation, skin inflammation, inflammatory bowel disease (IBD) including Crohn's disease and / or ulcerative colitis, irritable bowel syndrome, hepatitis, allergy, atopy, bone inflammation, rheumatoid arthritis, systemic lupus, Gougello-Sjögren's syndrome, Reiter's syndrome, poliomyelitis, dermatomyositis, thyroiditis, Graves' disease, Hashimoto's disease, type 1 diabetes, type 2 diabetes, Addison's disease, autoimmune hepatitis, celiac disease, Bielmer's disease, multiple sclerosis, myasthenia gravis, ophthalmitis, obesity-related inflammation, age-related mild inflammation, Blau syndrome, Alzheimer's disease, cardiovascular disease, atherosclerosis, metabolic syndrome, gingivitis, periodontitis, and combinations thereof.

12. The composition according to claim 1 or 2, wherein the inflammation is IBD.

13. The composition according to claim 12, wherein the IBD is Crohn's disease or ulcerative colitis.

14. The composition according to claim 12, wherein the individual is selected from the group consisting of infants, children, adolescents, adults, and the elderly.

15. The composition according to claim 12, wherein the at least one Staphylococcus carnosus strain is administered with a composition further comprising at least one component selected from the group consisting of prebiotics, amino acids, proteins, nucleotides, fish oil, non-marine omega-3 fatty acid sources, phytonutrients, antioxidants, and mixtures thereof.

16. The composition according to claim 4, wherein the disorder is selected from the group consisting of infectious tendencies, allergies, type 1 diabetes, insulin resistance, type 2 diabetes, celiac disease, peripheral and central obesity, obesity, necrotizing enterocolitis, inflammatory bowel disease, functional gastrointestinal disorders, and combinations thereof.

17. The composition according to claim 5, wherein the individual has damaged intestinal mucosa.

18. A unit dosage form of a composition comprising at least one Staphylococcus carnosus strain for treating at least one of inflammation, mucosal damage, or microbiome dysbiosis, comprising an amount of at least one Staphylococcus carnosus strain that produces one or more of tryptamine, indole, indolepropionic acid, 3-methylindole, indole-3-acetic acid, or indole-3-acetaldehyde, and is therapeutically or prophylactically effective against at least one of inflammation, mucosal damage, or microbiome dysbiosis.

19. A method for preparing a composition for treating at least one of inflammation, mucosal damage, or microbiome dysbiosis, comprising the step of adding at least one Staphylococcus carnosus strain producing one or more of tryptamine, indole, indolepropionic acid, 3-methylindole, indole-3-acetic acid, or indole-3-acetaldehyde to at least one component selected from the group consisting of prebiotics, amino acids, proteins, nucleotides, fish oil, non-marine omega-3 fatty acid sources, phytonutrients, antioxidants, and mixtures thereof.

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