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    • 专利摘要:
    Phascolarctobacterium faecium for use in the prevention and treatment of obesity and its comorbidities. The present invention relates to the P. faecium DSM 32890 strain, its cellular components, metabolites, and secreted molecules, its compositions and/or any of its combinations and its use to regulate appetite, and for the treatment and/or the prevention of overweight and/or obesity, and associated metabolic and immunological disorders; specifically, hyperglycemia, glucose intolerance, insulin resistance, dyslipidemia (hypertriglyceridemia, hypercholesterolemia), metabolic syndrome, diabetes, and intestinal and/or peripheral tissue inflammation. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2763874A1
    申请号:ES201831166
    申请日:2018-11-30
    公开日:2020-06-01
    发明作者:Herranz Yolanda Sanz；Almela Inmaculada López；DEL PULGAR VILLANUEVA EVA Mª GÓMEZ；Alfonso Benitez-Páez；Pérez Marina Romani
    申请人:Consejo Superior de Investigaciones Cientificas CSIC；
    IPC主号:
    专利说明:

    [0002] Phascolarctobacterium faecium for use in the prevention and treatment of obesity and its comorbidities
    [0004] The present invention falls within the pharmaceutical and food field. Specifically, the present invention relates to the deposit number strain DSM 32890, to its cellular components, metabolites, and secreted molecules, to its compositions and / or to any of its combinations. It particularly refers to the use of the P. faecium DSM 32890 strain to regulate appetite, and for the treatment and / or prevention of overweight and / or obesity, and associated metabolic and immunological disorders; specifically, hyperglycemia, glucose intolerance, insulin resistance, dyslipidemia (hypertriglyceridemia, hypercholesterolemia), metabolic syndrome, diabetes, hepatic steatosis, cardiovascular diseases, and intestinal and / or peripheral tissue inflammation.
    [0006] BACKGROUND OF THE INVENTION
    [0008] Obesity is one of the biggest public health problems due to its high prevalence and comorbidities, which greatly reduce the quality of life and increase the risk of mortality. These include, for example, dyslipidemia, metabolic syndrome, diabetes, cardiovascular disease, atherosclerosis, liver steatosis or fatty liver, and hypertension, as well as eating behavior disorders.
    [0010] Obesity occurs as a consequence of a prolonged imbalance, between intake and energy expenditure that leads to increased weight and body fat. The energy balance is regulated by neuroendocrine control systems in the long and short term. The key hormones in long-term control are insulin and leptin. Insulin is the most important hormone in the uptake of glucose and the regulation of the proper functioning of adipose tissue and the accumulation of triglycerides in it. In normal, insulin-sensitive adipose tissue, fat storage occurs here, in response to insulin and other hormones (leptin), by stimulating lipoprotein lipase and inhibiting lipolysis. However the Excessive accumulation of fatty acids in adipose tissue associated with obesity reduces insulin sensitivity, which promotes the accumulation of free fatty acids in the form of triglycerides in other organs and tissues (liver, muscle, etc.), and causes alterations in the production or sensitivity to leptin, and the increase in the synthesis of pro-inflammatory cytokines, which in turn carries an increased risk of development of associated diseases (metabolic syndrome, diabetes, cardiovascular diseases, etc.). Leptin is a hormone / adipokine synthesized primarily by adipose tissue, based on energy stores and in response to insulin. Leptin regulates energy homeostasis, acting at the level of the central and peripheral nervous system, decreasing energy intake and increasing energy expenditure. However, in obese subjects, peripheral leptin concentrations are abnormally high, resulting in resistance to it and lack of functionality. Among the short-term control systems are the intestinal hormones that are key to controlling intake at each meal and energy metabolism in various tissues. These hormones are released by enteroendocrine cells (EEC) in response to nutrients and nutrient metabolites, which reach the intestinal lumen where they are detected by specific receptors (eg, G protein-coupled receptors). These hormones include the cholecystokinin (CCK) secreted by I cells, mainly located in the proximal intestine, and the glucagon-like peptide 1 (GLP-1) and the YY peptide (PYY) secreted by L cells, mainly present in the distal region of the intestine. Once released, the intestinal hormones not only exert a direct effect on the distal organs (liver, white and brown adipose tissue) that allow controlling energy metabolism, but also act as mediators of central control of metabolism and eating behavior by neural and endocrine pathways through the gut-brain axis. Among the hormones mentioned, GLP-1 stands out, which suppresses appetite at the level of the hypothalamus and induces satiety, thus reducing food intake; improves glucose metabolism, by inducing insulin secretion in the pancreas and reducing glucagon synthesis; increases energy expenditure; and contributes to the reduction of body weight, liver steatosis and the risk of developing diabetes and cardiovascular diseases. PYY is more stable than GLP1 and, likewise, it acts by inducing satiety and thus contributing to the reduction of intake and body weight. It also reduces excessive food consumption by activating proopiomelanocortin. (POMC) and inhibition of neuropeptide Y (NPY) in the central nervous system. However, excessive consumption of energy-dense foods alters the synthesis of enteroendocrine hormones and their functionality, leading to increased intake and altered peripheral energy metabolism, exacerbating the obesity phenotype.
    [0012] Obesity is frequently associated with a low-grade state of chronic inflammation, implicated in metabolic complications, such as type 2 diabetes, cardiovascular disease, and fatty liver. Inflammation of white adipose tissue is considered a causal factor for these metabolic disorders and is characterized by a general increase in pro-inflammatory cells of the immune system such as M1 macrophages (classically activated) and IFNy-producing Th1 lymphocytes, T CD8 + and B Conversely, a reduction in anti-inflammatory M2 macrophages, Th2 lymphocytes, innate type 2 lymphoid cells (ILC2s) and, frequently, regulatory T cells (Tregs), which would control inflammation, is observed. Adipose tissue has been considered the main contributor to inflammation and metabolic dysfunction during obesity, but this phenomenon is now known to affect multiple organs, including the brain, muscles, liver, pancreas, and intestine. The most recent evidence suggests that the immune system associated with the intestine and the microorganisms that colonize and predominate it, as a consequence of exposure to hypercaloric and unhealthy diets, contribute to the metabolic inflammation associated with obesity and that the intestine may be the origin of metabolic inflammation.
    [0014] Among the factors involved in obesity, especially changes in lifestyle that lead to increased intake of high-energy-density foods and reduced physical activity and, therefore, energy expenditure, are considered the main causes of the obesity epidemic. Preventive and therapeutic strategies based on low calorie diets and increased physical activity represent the first option in the management of obesity and its complications, but they are generally limited in their long-term effectiveness. Therefore, adjuvant alternatives to changes in lifestyle are required to improve its effectiveness. On the other hand, pharmacological strategies, including those based for example on GPL-1 receptor agonists, present side effects in part due to which are consumed continuously when used to treat chronic pathologies. Furthermore, their effectiveness is limited because they are based on a single therapeutic target, without addressing the complexity of mechanisms that contribute to obesity and its complications.
    [0016] Obesity and its comorbidities (type 2 diabetes, dyslipidemia, cardiovascular disease, fatty liver, metabolic syndrome, etc.) have been associated with alterations in the composition and functions of the intestinal microbiota in observational studies in humans, suggesting that the intestinal microbiota could play a relevant role in these disorders. This hypothesis has been confirmed by transferring the microbiota from sick or healthy individuals to new subjects and observing that the latter acquired the donor phenotype. These experiments confirm that these alterations of the microbiota, in part due to hypercaloric diets, contribute to the development of obesity and its metabolic complications. This evidence has led to the development of intervention strategies on the intestinal ecosystem, such as the use of probiotics, as an alternative to improve the treatment and prevention of obesity. The products initially developed have been based on bacterial strains belonging to the Lactobacillus and Bifidobacterium genera , due to their history of safe use in food. It is now known, however, that other bacteria naturally present in a greater proportion in the human intestine and related to a small phenotype, could be more effective alternatives. Unlike pharmacological strategies, the use of commensal intestinal bacteria would have the advantage of being able to act through various mechanisms of action, regulating both the endocrine and immune systems and, a priori, without causing adverse effects. Regarding the possible beneficial properties of the genus Phascolarctobacterium, a study in rats published that pharmacological treatments used for diabetes (berberine and metformin) modified the microbiota and caused increases in different bacterial groups ( Allobaculum, Bacteriodes, Blautia, Butyricoccus, and Phascolarctobacterium) ; This led to speculation that this global modification of the microbiota could be part of the mode of action of anti-diabetics, but no direct evidence was provided. In a study of rats fed a high-fat diet and whether or not they underwent physical exercise, it was observed that the high-fat diet was related to a decrease in the Firmicutes edge and low ability to run with a reduction in Phascolarctobacterium; this led to speculating that changes in this microbial group might be partly responsible for the positive effects of physical exercise on fatty liver, but no direct evidence was provided. In no previous document the beneficial effects of specific species or strains of the genus Phascolarctobacterium on the alterations and pathologies object of the present invention are demonstrated.
    [0018] DESCRIPTION OF THE INVENTION
    [0020] The present invention relates to the deposit number strain DSM 32890, to its cellular components, metabolites, and secreted molecules, to its compositions and / or to any of its combinations, and to its use to regulate appetite, and for the treatment and / or the prevention of overweight and / or obesity, and associated metabolic and immunological disorders; specifically, hyperglycemia, glucose intolerance, insulin resistance, dyslipidemia (hypertriglyceridemia, hypercholesterolemia), metabolic syndrome, type-2 and gestational diabetes, liver steatosis and cardiovascular diseases, as well as inflammation intestinal and / or peripheral tissue and associated intestinal microbiota disorders.
    [0022] One of the main beneficial effects of the bacterium that is the subject of the patent, as well as of the derived products is in its ability to reduce the cellular and humoral mediators of inflammation, associated with obesity and that lead to metabolic dysfunction (for example , insulin resistance, metabolic syndrome and type 2 diabetes).
    [0024] As described in the examples, in vitro tests (Example 2) showed that the bacterium induces an anti-inflammatory response in peripheral blood mononuclear cells (PBMCs) since it increases the production of the anti-inflammatory cytokine IL-4 with respect to the pro-inflammatory IFNy and decreases the levels of classic monocytes (CD14 ++ CD16-) with respect to the effects induced by the lipoplisaccharide LPS, inducer of inflammation in obesity (Table 1). Said anti-inflammatory effect can contribute to improve insulin resistance and glucose intolerance caused by the pro-inflammatory state of obesity to a greater extent than other intestinal bacteria (Example 2).
    [0025] A fundamental aspect of the invention is the ability of P. faecium to reduce inflammation and protect the intestinal mucosa, reducing the risk of systemic inflammation associated with obesity and metabolic complications in vivo. Specifically, in a diet-induced obesity model (Example 3), P. faecium reduces intestinal inflammation associated with obesity, acting on both the innate and acquired immune systems. For example, this bacterium works by reducing the population of group 1 innate lymphoid cells (Innate Lymphoid Cells, ILC1) in the epithelium, which are increased in obese individuals and promote intestinal inflammation by IFN production and (Example 3; Figure 4a). The production of IFN and by these cells triggers the polarization of macrophages towards those of the M1 type (pro-inflammatory phenotype), closely involved in the inflammation associated with obesity; however, the bacteria is able to reverse the effect of diet by increasing the levels of M2-type macrophages, thus inducing an anti-inflammatory response (Example 3; Figure 4b) characterized by normalization of the ratio of M1 / M2 cells (Example 3 ; Figure 4c) in the lamina propria of the intestine. Furthermore, P. faecium reduces the inflammatory tone associated with obesity, inducing a Th2-type response and the production of Treg cells (Example 3; Figures 4d and 4e, respectively). This response has been demonstrated by measuring the levels of Gata3 as a mediating transcription factor of a Th2 response and the levels of regulatory T cells with the CD25 and FoxP3 markers. The increase in Gata3 also indicates the possible increase in the population of ILC2 cells, characterized by producing Th2-type cytokines, which exert an anti-inflammatory effect in the context of obesity. The Gata3 transcription factor, in addition to being essential for the development of ILC2 from hematopoietic stem cells (HSC), is also abundant in a subset of ILC3 associated with the mucosa. The increase in the latter type of cells (ILC3) in the lamina propria may also contribute to increasing the protection of the intestinal mucosa and the barrier function, which is altered in obesity and may contribute to systemic inflammation. P. faecium also reduces intraepithelial lymphocyte (IEL) levels and normalizes the ratio of natural / induced IELs (Figure 5), altered in obesity. The IELs are located between the epithelial cells of the intestinal tract and, therefore, they play an essential role in the control of the integrity of the epithelium and the alteration of its phenotype can contribute to alterations in intestinal permeability and activation of the inflammatory tone characteristic of the obesity.
    [0026] The patent bacterium also has the ability to modulate the production of gastrointestinal hormones involved in glucose metabolism and appetite regulation, such as GLP-1 and PYY. P. faecium restores PYY and GLP-1 precursor expression in the small intestine, which is altered in mice with obesity induced by a hypercaloric diet. These hormones are directly responsible for the recovery of glucose homeostasis, the reduction of body weight and intake by facilitating the transmission of satiety signals to brain regions involved in the control of intake and improving the functioning of peripheral tissues involved in the energy metabolism (for example, insulin secretion in the pancreas and energy expenditure in adipose tissue). These effects would reduce the alterations in dietary intake and lipid and glucose metabolism, frequently associated with obesity, and which constitute a risk factor that precedes the development of metabolic syndrome and type 2 and gestational diabetes and cardiovascular pathologies.
    [0028] Therefore, in one aspect, the present invention relates to strain P. faecium with deposit number DSM 32890, hereafter "strain of the invention" or "strain DSM 32890 or" strain P. faecium DSM 32890.
    [0030] P. faecium was isolated from human faeces. The strain was deposited by the Higher Council for Scientific Research (CSIC) on October 9, 2018 under the Budapest Treaty at the Deutsche Sammlung von Mikroorganismen und Zellkulture as the International Depository Authority (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures , InhoffenstraBe 7B, 38124 Braunschweig, GERMANY). The assigned deposit number was DSM 32890.
    [0032] The scientific classification of the strain of the invention according to the NCBI database is Domain: Bacteria; Edge: Firmicutes; Class: Negativicutes; Order: Acidaminococcales; Family: Acidaminococcaceae; Genus: Phascolarctobacterium; Species: faecium.
    [0034] It is an abundant bacterium in the intestinal tract of healthy humans, Gram-negative anaerobia of bacillary morphology, non-spore-forming and immobile; They are usually short bacilli, although their size can vary depending on the growth phase and lengthen. The colonies are transparent and of inconsistent texture.
    [0035] It grows at 37 ° C in strict anaerobiosis. The bacterium requires succinate, as a carbon source, for its growth and from this substrate it produces propionate.
    [0037] Another aspect of the present invention relates to a strain derived from the P. faecium DSM 32890 strain, where said strain maintains or improves the capabilities described throughout the present invention. The derived microorganism can be produced naturally or intentionally, by mutagenesis methods known in the state of the art, such as, but not limited to, the growth of the original microorganism in the presence of mutagenic or stress causing agents, or by engineering. genetics aimed at modifying specific genes. According to a preferred embodiment, the strain derived from the P. faecium DSM 32890 strain is a genetically modified mutant. The terms mutant strain or derived strain can be used interchangeably.
    [0039] The P. faecium DSM 32890 strain or any mutant or derivative thereof can be used in any way that exerts the described effects. According to a preferred embodiment of the present invention, the strain P. faecium DSM 32890 is in the form of viable cells (cultivable or non-cultivable), or according to another preferred embodiment of the invention the strain is in the form of non-viable cells ("dead" cells). "Inactivated by any technique known in the state of the art such as, but not limited to, heat, freezing or ultraviolet radiation).
    [0041] Another aspect of the present invention refers to the cellular components, metabolites, secreted molecules or any of their combinations, obtained from the strain of the invention, or from a combination of microorganisms comprising at least one strain of the invention.
    [0043] The cellular components of the bacterium could include components of the cell wall (such as, but not limited to, peptidoglycan), nucleic acids, membrane components, or others such as proteins, lipids, and carbohydrates and their combinations, such as lipoproteins, glycolipids, or glycoproteins. Metabolites include any molecule produced or modified by the bacterium as a consequence of its metabolic activity during its growth, its use in technological processes (for example, but not limited to, food or drug manufacturing processes), during product storage, or during gastrointestinal transit. Examples of these metabolites are, but are not limited to, organic and inorganic acids, proteins, peptides, amino acids, enzymes, lipids, carbohydrates, lipoproteins, glycolipids, glycoproteins, vitamins, salts, metals, or nucleic acids. Secreted molecules include any molecule exported or released abroad by the bacterium during its growth, its use in technological processes (for example, food or drug manufacturing), product storage, or gastrointestinal transit. Examples of these molecules are, but are not limited to, organic and inorganic acids, proteins, peptides, amino acids, enzymes, lipids, carbohydrates, lipoproteins, glycolipids, glycoproteins, vitamins, salts, metals, or nucleic acids.
    [0045] Another aspect of the present invention relates to a composition, hereinafter "composition of the invention", comprising the strain of the invention and / or the cellular components, metabolites, secreted molecules of the strain of the invention or any of their combinations.
    [0047] The composition, defined generally, is a set of components that is formed by at least the strain of the invention in any concentration; or at least by the cellular components, metabolites, secreted molecules of the strain of the invention or any of its combinations; or by a combination thereof.
    [0049] In a preferred embodiment, the composition of the invention has a concentration of the strain of the invention of between 103 and 1014 colony forming units (cfu) per gram or milliliter of final composition.
    [0051] In another particular embodiment, the composition of the invention may further comprise at least one additional microorganism other than the strain of the invention and / or its secreted cellular components, metabolites or molecules, or any combination thereof. For example, but not limited to, the additional microorganism that may be part of said composition is selected from at least one of the following groups:
    [0053] - at least one lactic bacteria or bifidobacterium of intestinal, food or environmental origin. Lactic acid bacteria is selected from the list consisting of, but not limited to, a bacterium of the genus Bifidobacterium, Lactobacillus, Lactococcus, Enterococcus, Propionibacterium, Leuconostoc, Weissella, Pediococcus or Streptococcus;
    [0054] - at least one strain of another species of the genus Bacteroides or of the species Bacteroides uniformis;
    [0055] - at least one strain of other phylogenetic groups, genera or species of prokaryotes of intestinal, food or environmental origin, such as but not limited to Archaea, Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Verrucomicrobia, Fusobacteria, Metanobacteria, Spirochaetes, Fibrobacteres, Deferribacteres, Deinococcus, Thermus, Cyanobacteria, Methanobrevibacterium, Peptostreptococcus, Ruminococcus, Coprococcus, Subdolingranulum, Dorea, Bulleidia, Anaerofustis, Bacteria, Catenibacterium, Dialister, Anaerotrcoccus, Anaerotruncus Eubacterium, Akkermansia, Bacillus, Butyrivibrio, Clostridium or Mycobacterium;
    [0056] - at least one strain of fungus or yeast such as, but not limited to, belonging to the genus Saccharomyces, Candida, Pichia, Debaryomyces, Torulopsis, Aspergillus, Rhizopus, Mucor or Penicillium .
    [0058] Said additional microorganism may be a strain of the same species or of a different species or taxonomic group of microorganisms from that corresponding to the strain of the invention. The cells that comprise the composition can be non-viable or viable and be in any stage of the state of development or growth (latent, exponential, stationary, etc.), regardless of the morphology it presents. In a particular embodiment, said additional microorganism comprises at least one intestinal bacterium or a lactic acid bacterium.
    [0060] Optionally, in another particular embodiment, the composition of the invention may further comprise at least one bioactive component (active substance, active ingredient or therapeutic agent), such as, for example, other components of food, plant products and / or drugs.
    [0062] The term "bioactive component" refers to a compound with biological activity in the scope of the patent that can improve or complement the activity of the DSM 32890 strain, including ingredients or components of food (for example and without limitation: acids polyunsaturated fats, conjugated linoleic acid, prebiotics, fiber, Guar gum, glucomannan, chitosan, copper, calcium, etc.), other probiotics, plants, extracts or components of plants and drugs.
    [0064] In a particular embodiment, the composition of the invention is a pharmaceutical composition. The pharmaceutical composition is a set of components that is formed by at least the strain of the invention in any concentration; or at least by the cellular components, metabolites, secreted molecules of the strain of the invention or any of their combinations, which has at least one application in the improvement of the physical or physiological or psychological well-being of a subject, which implies an improvement of the state general health or reduced risk of disease. Said pharmaceutical composition may be a medicine.
    [0066] The term "medicine" has a more limited meaning than the meaning of "pharmaceutical composition", as defined in the present invention, since the medicine necessarily implies a preventive or therapeutic effect. The medicine to which the present invention refers can be for human or veterinary use. The "medicine for human use" is any substance or combination of substances that is presented as possessing properties for the treatment or prevention of diseases in humans or that can be used in humans or administered to humans in order to restore, correct or to modify the physiological functions exerting a pharmacological, immunological or metabolic action, or to establish a medical diagnosis. "Veterinary medicine" is any substance or combination of substances that is presented as having curative or preventive properties with respect to animal diseases or that can be administered to the animal in order to restore, correct or modify its physiological functions by exercising a pharmacological, immunological or metabolic action, or to establish a veterinary diagnosis. "Veterinary drugs" are also considered "veterinary drugs" prepared to be incorporated into a feed.
    [0068] In addition to the requirement for therapeutic efficacy where such a pharmaceutical composition may necessitate the use of other therapeutic agents, there may be additional fundamental reasons that strongly compel or recommend the use of a combination of a compound of the invention and a biactive component, where a said bioactive component is attributed an appropriate activity to constitute a medicine. Said compound of the invention obviously refers to the strain of the invention, or to the strain derived from it, or to the cellular components, metabolites, secreted molecules or any of their combinations, obtained from the strain of the invention.
    [0070] In a particular embodiment, the pharmaceutical composition further comprises at least one pharmacologically acceptable vehicle and / or excipient.
    [0072] The "vehicle" or carrier is preferably an inert substance. The function of the vehicle is to facilitate the incorporation of other compounds, to allow for better dosing and administration, or to give consistency and shape to the pharmaceutical composition. Therefore, the vehicle is a substance which it is used in the medicine to dilute any of the components of the pharmaceutical composition of the present invention up to a determined volume or weight, or even without diluting said components it is capable of allowing better dosage and administration or giving consistency and shape to the medicine When the presentation form is liquid, the pharmaceutically acceptable vehicle is the diluent.
    [0074] The term "excipient" refers to a substance that assists in the absorption of any of the components of the composition of the present invention, stabilizes said components or helps in the preparation of the pharmaceutical composition in the sense of giving it consistency or providing flavors that Thus, the excipients could have the function of keeping the components together, such as starches, sugars or cellulose, sweetening function, dye function, protection function of the medicine, for example, to isolate it from the air and / or or humidity, filling function of a pill, capsule or any other form of presentation such as, for example, dibasic calcium phosphate, a disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph. Therefore, the term "excipient" is defined as that matter that, included in the forms ga lenicas, it is added to the active ingredients or their associations to enable their preparation and stability, modify their organoleptic properties or determine the physico-chemical properties of the pharmaceutical composition and its bioavailability. The "pharmaceutically acceptable" excipient must allow the activity of the compounds of the pharmaceutical composition, that is, be compatible with these components.
    [0076] Furthermore, as understood by the person skilled in the art, the excipient and the vehicle must be pharmacologically acceptable, that is, that the excipient and the vehicle are allowed and evaluated so as not to cause harm to the organisms to which it is administered.
    [0078] The pharmaceutical composition or medicament can be presented under any clinically permitted administration form and in a therapeutically effective amount. For example, it may be in a form adapted for oral, sublingual, nasal, intracathecal, bronchial, lymphatic, rectal, transdermal, inhaled, or parenteral administration, preferably in a form adapted for oral administration. The pharmaceutical composition of the invention can be formulated in solid, semi-solid, liquid or gaseous forms, such as tablet, capsule, powder, granule, ointment, solution, suppository, injection, inhalant, gel, microsphere or aerosol. The form adapted for oral administration is selected from the list comprising, but not limited to, drops, syrup, herbal tea, elixir, suspension, extemporaneous suspension, drinkable vial, tablet, capsule, granule, seal, pill, tablet, pill, troche or lyophilized. In a particular embodiment, the composition of the invention is presented in a form adapted for oral, sublingual, nasal, bronchial, lymphatic, rectal, transdermal, inhaled or parenteral administration.
    [0080] In a more particular embodiment, the composition of the invention is presented in a form adapted for oral administration. The form adapted for oral administration refers to a physical state that can allow oral administration. Said form adapted for oral administration is selected from the list comprising, but not limited to, drops, syrup, herbal tea, elixir, suspension, extemporaneous suspension, drinkable vial, tablet, capsule, granule, seal, pill, tablet, pill, troche or lyophilized.
    [0082] The "galenic form" or "pharmaceutical form" is the disposition to which the active ingredients and excipients are adapted to constitute a medicine. It is defined by the combination of the way in which the pharmaceutical composition is presented by the manufacturer and the way in which it is administered.
    [0084] In the present invention, the term "therapeutically effective amount" refers to that amount of the component of the pharmaceutical composition that when administered to a mammal, preferably a human, is sufficient to produce the prevention and / or treatment, as defined below, of a disease or pathological condition of interest in the mammal, preferably a human. The therapeutically effective amount will vary, for example, according to the activity of the strain of the invention; of the cellular components, metabolites, secreted molecules or any of their combinations, in any form of presentation; the therapeutically effective amount will also vary according to the metabolic stability and duration of action of the compound; the age, body weight, general health, sex and diet of the patient; the mode and time of administration; excretion rate, drug combination; the severity of the particular disorder or disease condition; and the subject undergoing therapy, but may be determined by a specialist in the art based on his or her own knowledge and description.
    [0086] As an alternative to the pharmaceutical composition, the composition of the invention can also be a nutritional composition.
    [0088] The term "nutritional composition" of the present invention refers to that food that, independently of providing nutrients to the subject who takes it, beneficially affects one or more functions of the organism, so as to provide a better state of health and well-being. As a consequence, said nutritional composition can be used for the prevention and / or treatment of a disease or the causative factor of a disease. Therefore, the term "nutritional composition" of the present invention can be used as a synonym for functional food or food for specific nutritional purposes or medicinal food.
    [0090] In a particular embodiment, the nutritional composition is a food, a supplement, a nutraceutical, a probiotic, or a symbiotic.
    [0092] In a more particular embodiment, the food is selected from the list consisting of a dairy product, a vegetable product, a meat product, a snack, chocolate, beverage or baby food. The dairy product is selected from the list consisting of, a product derived from fermented milk (for example, but not limited to, yogurt or cheese) or unfermented (for example, but not limited to, ice cream, butter, margarine, dairy serum). The plant product is, for example, but not limited to, a cereal in any form, fermented or unfermented. The beverage may be, for example, but not limited to, any fruit juice or unfermented milk.
    [0094] The term "supplement", synonymous with any of the terms "dietary supplement", "nutritional supplement"; or "food supplement" is a "food ingredient" intended to supplement food. Some examples of dietary supplements include, but are not limited to, vitamins, minerals, botanicals, amino acids, and food components such as enzymes and glandular extracts. They are not presented as substitutes for a conventional food or as a single component of a meal or diet but rather as a supplement to the diet.
    [0096] The term "nutraceutical" as used in the present invention refers to substances isolated from a food and used in dosages that have a beneficial effect on health.
    [0098] The term "probiotic" as used in the present invention refers to live microorganisms that when supplied in adequate amounts promote health benefits of the host organism.
    [0100] The term "symbiotic" as used in the present invention refers to those foods that contain a mixture of prebiotics and probiotics. As a general rule, they contain a prebiotic component that favors growth and / or metabolic activity and, ultimately, the effect of the probiotic with which it is combined, such as, for example, and without limitation, may be the association of fructooligosaccharides or galactooligosaccharides with bifidobacteria.
    [0102] Another aspect of the present invention relates to the use of the strain of the invention, or the components derived from it, or the composition of the invention, for the manufacture of a medicine, a nutritional composition or a food.
    [0104] Another aspect of the present invention relates to the P. faecium DSM 32890 strain, a cellular component, metabolite, secreted molecule or any of its combinations obtained from the strain of the invention, or the composition of the invention, for use as a medicine. The term medicament has been previously defined, and is applicable to the present inventive aspect. As explained in previous paragraphs, this medicine can be a pharmaceutical composition or a nutritional composition.
    [0106] In another aspect, the present invention relates to the strain of the invention, a cellular component, metabolite, secreted molecule or any of its combinations obtained from the strain of the invention, or the composition of the invention, for use in prevention. and / or the treatment of overweight and / or obesity, or diseases associated with it.
    [0108] The term "treatment", as understood in the present invention, refers to combating the effects caused by a disease or pathological condition of interest in a subject (preferably a mammal, and more preferably a human) that includes:
    [0109] (i) inhibit the disease or pathological condition, that is, stop its development; (ii) alleviating the disease or pathological condition, that is, causing the regression of the disease or pathological condition or its symptoms;
    [0110] (iii) stabilize the disease or pathological condition.
    [0112] The term "prevention" as understood in the present invention consists in preventing the appearance of the disease, that is, preventing the disease or pathological condition from occurring in a subject (preferably mammalian, and more preferably a human), in particularly, when said subject is predisposed by the pathological condition.
    [0114] The term "overweight" refers to a pathology characterized in that the subject has a body mass index (BMI) equal to or greater than 25. BMI is a measure of association between the weight and height of an individual. The BMI has the following formula for its calculation: the weight of the subject divided by the height of the subject squared (Kg / m2). Overweight is characterized by a BMI between> 25 to <30. This calculation method is only valid for people over 18 years of age, and as the expert in the field knows, it is not applicable to minors if a correction factor is not applied. . Determination of said correction factor is standard practice for the person skilled in the art.
    [0115] The term "obesity" refers to a pathology characterized in that the subject has a BMI equal to or greater than 30. Obesity is classified at different levels, considering that subjects with a BMI> 40 suffer from morbid obesity. Other parameters used to determine whether an individual has central obesity are absolute waist circumference (the subject is obese when it is> 102 cm in men [central obesity] and> 88 cm in women) or the waist-hip ratio (the subject has obesity when it is> 0.9 for men and> 0.85 for women). An alternative way to determine obesity is to measure the percentage of body fat (the subject is obese when they have approximately> 25% body fat in a man and approximately> 30% body fat in women).
    [0117] In the present invention, "diseases associated with overweight and / or obesity" are understood as those diseases that are a consequence of the overweight or obesity suffered by the subject. Examples of diseases associated with overweight and / or obesity include, without limitation, cardiovascular diseases (such as heart disease, stroke, etc.), metabolic syndrome, diabetes (in particular, type II diabetes), hyperglycemia, insulin resistance, cancer (examples of cancer include, but are not limited to, endometrium, breast, ovaries, prostate, liver, gallbladder, kidneys, and colon), hypertension, dyslipidemia, hypolipidemia, galactosemia, phenylketonuria, sitysterolemia, hyperthyroidism, and hypothyroidism.
    [0119] Thus, in a particular embodiment, diseases associated with overweight and / or obesity are selected from the list consisting of cardiovascular diseases, metabolic syndrome, diabetes, hyperglycemia, insulin resistance, cancer, hypertension, dyslipidemia, hypolipidemia, galactosemia, phenylketonuria , sitosterolemia, hyperthyroidism and hypothyroidism.
    [0121] In the present invention, the term "cardiovascular disease" or "heart disease" refers to those diseases affecting the heart and blood vessels including, but not limited to, atherosclerosis, aneurysm, angina, stroke, cerebrovascular disease, heart failure congestive, coronary artery disease, acute myocardial infarction and peripheral vascular disease.
    [0122] In the present invention, the term "metabolic syndrome" refers to disease comprising a group of conditions that put the individual at risk of developing heart disease and type 2 diabetes. Examples of these conditions include, but are not limited to, high blood pressure. , high blood glucose, high blood levels of triglycerides, low blood levels of HDL and excess fat around the waist.Chronic inflammation and impaired lipid metabolism (dyslipidemia) and glucose are risk factors for cardiovascular pathologies and therefore, its treatment and prevention can prevent the development of this other group of pathologies
    [0124] In the present invention, "diabetes" is understood to be a disease characterized by having high levels of glucose in the blood because the body does not produce insulin or the cells are not able to use insulin. Insulin is a hormone that helps glucose entering cells for energy Over time, a high level of glucose in the blood can cause serious problems with the heart, eyes, kidneys, nerves, gums, and teeth.
    [0126] In the present invention, the term "hyperglycemia" refers to an excessive amount of glucose in the blood. Methods for measuring the amount of glucose in the blood, as well as the glucose value from which an excess of glucose is considered to be in blood, are widely known in the state of the art, and their use is routine practice for the person skilled in the art.
    [0128] In the present invention, "insulin resistance" or "insulin resistance" is understood to be the condition in which the tissues have a decreased response to dispose of circulating glucose to the action of insulin; especially the liver, skeletal muscle, adipose tissue, and the brain. This alteration, together with the deficiency of insulin production by the pancreas, can lead after some time to the development of type 2 diabetes mellitus.
    [0130] In the present invention, "cancer" is understood to be a disease in which there are abnormal cells that multiply uncontrollably and can invade nearby tissues. Within the term cancer, tumors are included, these being an abnormal mass of tissue that appears when cells multiply more than they should or are not destroyed at the appropriate time, and can be classified as benign (they do not invade nearby tissue does not spread to other parts of the body) or malignant (they invade nearby tissue and spread to other parts of the body).
    [0132] In the present invention, "hypertension" is understood to mean the elevation of blood pressure levels continuously or sustained with respect to a normal blood pressure (The maximum systolic blood pressure (maximum) levels are between 120 129 mmHg, and those of diastolic (minimum) between 80 and 84 mmHg).
    [0134] In the present invention, "dyslipidemia" is understood to mean the elevation of plasma cholesterol or triglyceride concentrations, or the decrease of high-density liprotein concentrations that contribute to the development of atherosclerosis. "Hypolipidemia" is understood to mean a decrease in the plasma concentration of lipoproteins, and is defined as a concentration of total cholesterol (CT) <120 mg / dL (<3.1 mmol / L) or cholesterol associated with lipoprotein of low density (LDL) <50 mg / dL (<1.3 mmol / L).
    [0136] In the present invention, “galactosemia” is understood as a hereditary disease characterized in that the individual is unable to use simple sugar galactose, which causes an accumulation of it within the organism, causing lesions in the liver and the central nervous system.
    [0138] In the present invention, "phenylketonuria", also known as PKU, is understood to mean the congenital alteration of the metabolism caused by the lack of the enzyme phenylalanine hydroxylase, which results in the inability to metabolize the amino acid tyrosine from phenylalanine in the liver.
    [0140] In the present invention, "sitosterolemia" is understood to be a rare autosomal recessive sterol storage disease characterized by the accumulation of phytosterols in blood and tissues. The clinical manifestations include xanthomas, arthralgia, and premature atherosclerosis. Hematologic features include hemolytic anemia with stomatocytosis and macrochrombocytopenia. The disease is caused by homozygous or compound heterozygous mutations in the genes ABCG5 (2p21) and ABCG8 (2p21).
    [0141] In the present invention, “hyperthyroidism” is understood as that disease in which the thyroid gland produces too much thyroid hormone, while “hypothyroidism” is understood as that disease in which the thyroid gland does not produce enough thyroid hormone to satisfy the needs of the Body.
    [0143] In another aspect, the present invention relates to the non-therapeutic use of the strain of the invention, a cellular component, metabolite, secreted molecule or any of its combinations obtained from the strain of the invention, or the composition of the invention, for regulation of appetite and / or food intake.
    [0145] Throughout the description and claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge in part from the description and in part from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
    [0147] BRIEF DESCRIPTION OF THE FIGURES
    [0149] Figure 1. Effect of the administration of the P. faecium strain (1x107-8 cfu / day) to obese C57BL / 6 mice (n = 10 / group) for 14 weeks, on the gain of body weight. (a) Body weight weekly. (b) Body weight gain after 14 weeks of treatment. Data are expressed in grams with means and standard error. Statistically significant differences were established by applying a one-way ANOVA followed by the Tukey test (p <0.05). CD, control diet; HFHSD, diet rich in fat and sugars; HFHSD + P.faecium, with a diet rich in fat and P.faecium sugars .
    [0151] Figure 2. Effect of the administration of the P. faecium strain (1x107-8 cfu / day) to obese C57BL / 6 mice (n = 10 / group) for 14 weeks, on basal glycemia and glucose tolerance. (a) Fasting blood glucose levels (mg / dL) at week 8 and 10. (b) Glucose tolerance test, blood glucose was measured at 15, 30, 60 and 120 minutes after having administered an oral overload. glucose (2 g / Kg). The area under the curve (Area Under the Curve, AUC) is shown for the glucose tolerance test results. Data are represented by means and standard error. The Statistically significant differences were established by applying a one-way ANOVA followed by the Tukey test (p <0.05). CD, control diet; HFHSD, diet rich in fat and sugars; HFHSD + P. faecium, with a diet rich in fat and P. faecium sugars .
    [0153] Figure 3. Effect of the administration of the P. faecium strain (1x107-8 cfu / day) to obese C57BL / 6 mice (n = 10 / group) for 14 weeks on intake. (a) Relative intake (kcal / day) per animal at weeks 3, 6, 9 and 12. (b) Daily intake (kcal) per animal at week 12 of treatment. (c) Size of white adipose tissue (grams). Data are represented by means and standard error. Statistically significant differences were established by applying a one-way ANOVA followed by the Tukey test (p <0.05). CD, control diet; HFHSD, diet rich in fat and sugars.
    [0155] Figure 4. Effect of administration of the P. faecium strain (1x107-8 cfu / day) to obese C57BL / 6 mice (n = 10 / group) for 14 weeks on inflammation. (a) Percentage of ILC1 cells in the epithelium, (b) percentage of macrophages with M2 phenotype (Anti-inflammatory), (c) ratio of M1 / M2 macrophages (Pro-inflammatory / Anti-inflammatory), (d) Average fluorescence intensity ( MFI) of the transcription factor Gata3 and (e) percentage of regulatory T lymphocytes (Treg). Data are represented by means and standard error. Statistically significant differences were established by applying a one-way ANOVA followed by the Tukey test (p <0.05). CD, control diet; HFHSD, diet rich in fat and sugars.
    [0157] Figure 5. Effect of the administration of the P. faecium strain (1x107-8 cfu / day) to obese C57BL / 6 mice (n = 10 / group) for 14 weeks on intraepithelial lymphocytes. (a) Percentage of induced intraepithelial lymphocytes (indicidal IEL) and (b) ratio of natural and induced intraepithelial lymphocytes (nat / ind IEL). Data are represented by means and standard error. Statistically significant differences were established by applying a one-way ANOVA followed by the Tukey test (p <0.05). CD, control diet; HFHSD, diet rich in fat and sugars.
    [0159] EXAMPLES
    [0161] Example 1. Isolation and identification of the bacterial strain P. faecium G104 ( P.
    [0162] faecium DSM 32890)
    [0164] Different intestinal bacteria were isolated from faeces from healthy volunteers. 1.25 grams of faeces were used and diluted in 10 mM phosphate buffer with 0.05% cysteine (1:10 dilution) containing a concentration of 130 mM NaCl (PBS) and homogenized in a stomacher Lab-Blender 400 (Seward Medical, London, 35 UK). Said dilution was inoculated in 37.5 mL of Intestinal Bacteria Medium (MBI) whose composition is based on the media recommended in previous publications (Gibson, GR, et al., Appl. Environ. Microbiol., 54 (1 1): 2750-5, 1988; Lesmes, U et al., J. Agric. Food Chem., 56: 5415-5421, 2008), with some modifications designed by the inventors:
    [0165] ■ Main ingredients: distilled water (1,600 mL), peptone water (4 g), NaHCO3 (4 g), CaCl2 (0.02 g), pectin (4 g), xylan (4 g), wheat bran extract (4 g), arabinogalactans (2 g), gum arabic (2 g), starch (10 g), casein (6 g), inulin (2 g) and NaCl (0.2 g). Autoclaved at 121 ° C for 60 minutes and allowed to cool until the next day.
    [0166] ■ Mucin solution: Mucin (8 g) and distilled water (200 mL). Autoclaved 20 minutes.
    [0167] ■ Salts and vitamins: distilled water (100 mL), K2HPO4 (0.08 g), KH2PO4 (0.08 g), MgSO4 (0.02 g), hemin (0.01 g) and menadione (0.002 g)
    [0168] ■ Cysteine solution: L-cysteine-HCl (1 g), distilled water (100 mL)
    [0170] The mixture of vitamins and salts and the cysteine solution were combined and 6M KOH was added until the final solution turned translucent brown and was filter sterilized. The final MBI was obtained by mixing the main ingredients, the mucin solution, the salts and vitamins, and the cysteine solution, completing a volume of 2 L under sterile conditions.
    [0172] The 50 mL of faeces diluted in MBI medium were fermented for 24 hours in an anaerobic chamber (Whitley DG250 Workstation, Don Whitley Scientific) with stirring keeping the pH between 6.9-7.0. The 24-hour fermented MBI medium itself was filtered (pore size 0.22 pm) and used as a supplement to Fastidious Anaerobe Agar (FAA) medium agar plates with 0.5% defibrillated blood, in which seeding serial dilutions of the fermented stool (0.1 mL of inoculum from each serial dilution on each plate). This MBI medium supplement Fermented contains substrates produced by the intestinal microbiota, being a medium enriched with nutrients present in the intestinal ecosystem that allows a better recovery of native bacteria under laboratory conditions. The seeded plates were incubated 72 hours at 37 ° C and in an anaerobic chamber.
    [0174] Phascolactobacterium faecium DSM 32890 was isolated from the colonies that grew after 72 hours in plate. Its identification was carried out by sequencing the 16S rRNA gene (1.26 Kb) using the 27f primers (5'-AGAGTTT GATCCTGGCTCAG-3 '(SEQ ID NO: 1)) and 1401r (5'-CGGTGTGTACAAGACCC-3 '(SEQ ID NO: 2)). Reactions after DNA amplification were purified with the Illustra GFX PCR DNA and Gel Band Purification Kits kit (GE Healthcare) and sequenced by Sanger technology on an ABI 3730XL sequencer (Stabvida-Caparica-Portugal). By comparison of the sequences obtained with the NCBI database and the BLASTn algorithm, the identification of the isolated strain (G104) with the species Phascolactobacterium faecium strain ACM 3679 (partial sequence, 16S ribosomal RNA) with a percentage identity was obtained. 99%. The new strain Phascolactobacterium faecium G104 was deposited in the German Type Culture Collection, corresponding to the number DSM 32890.
    [0176] 16S sequence used for identity identification by Blastn algorithm using oligos 27F and 1401r for its sequencing was as follows:
    [0178] > 16S G104 partial sequence (SEQ ID NO: 3)
    [0179] TCCGACTTCACGCAGGCGGGTTGCAGCCTGCGATCCGAACTGAGATCGGGTTTCTGGGGTTTGC TCTGCCTCG CG G CTTCG CTTCCCTCTG TTTCCG ACCA TTG TAG TA CG TG TG TAG CCCAA GA CA T AAG GGG CATGATGACTTGACGTCATCCCCGCCTTCCTCCAG GTTGTCCCTG GCAGTCTCCCATG AGTCCCCAACTTTACTTGCTGGTAACATAGGATAGGGGTTGCGCTCGTTGCGGGACTTAACCCA ACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTTTTCTTGTCCCCGAAGGGAAAT CTCTA TCTCTA GAG CG TTCAATCAATG TCAAGCCTTGG TAAGGTTCTTCG CGTTG CGTCG AATT A AA CCA CATA CTCCACCGCTTG TGCG GGCCCCCG TCAATTCCTTTG AG TTTCAACCTTG CGG CC GTACTCCCCAGGCGGGGTACTTATTGCGTTAACTCCGGCACAGAAGGGGTCGATACCTCCTACA CCTAGTACCCATCGTTTACG GCCAGG ACTACCG GG GTATCTAATCCCGTTCG CTACCCTG GCTT TCGCATCTCAG CGTCAGACACAGTCCAGAAAGGCGCCTTCGCCACTGGTGTTCCTCCCAATATC TA CG CA TTTCACCG CTA CA CTG GGAA TTCCCCTTCA CA CTTC CAGCGCCATACGGGGTTGAGCCCCGCATTTTCACGCTCGACTTATTAAGCCG CCTACATGCTCT TTACGCCCAATAATTCCGGACAACGCTCGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGT TAG CCG TG G CTTCCTCG TTTACTACCG TCA TTG CA A TG CATTG TTCACA CA CTG CACG TGCTs TC ATAAACAACAGAGCTTTACAGACCGAAATCCTTCATCACTCACGCGGCGTTGCTCCGTCAGACT TGCTs TCCA TTG CG G AAGATTCCCCACTG CTGCCTCCCGTAG GAGTTTGG GCCGTGTCTCAGTCC CA ATG TG G CCG TTCATCCTCTCAGACCGGCTACTG ATCATCGCCTTG GTAG TCCGTTACACTAC CAACTAGCTAATCAGACGCAGG CCCATCCTTTAG CGATAGCTTACTTGTAG AG GCCATCTTTCT TCATCCTG CCATGCGG CACG ATGATCACATCCG GTATTAGCACTCCTTTCG GAATGTTG TCCCC GTCTAAAGGGCAGGTTGCCTACGCGTTACTCACCCGTTCGCCACTAAGAATTCTACCGAAATAA
    [0181] The specific growth of this strain was optimized for future tests using the medium recommended by the DSMZ culture collection (Medium n ° 104b (PY succinate 8 g / L)).
    [0183] For 1L medium, peptone trypticase 5.0 g, meat extract 5.0 g, yeast extract 10.0 g, K2HPO42.00 g, Tween 80 1.00 mL, salt solution (see below) were mixed 40.0 mL, 1 mg resazurin solution, L-Cysteine-HCl x H2O 0.5 g, sodium succinate 8.0 g, distilled water 950.0 mL, hemin solution (see below) 10.00 ml and vitamin K1 solution (see bottom) 0.20 mL.
    [0184] • Salt solution: CaCl2 x 2 H2O 0.25 g, MgSO4 x 7 H2O 0.50 g, K2HPO41.00 g, KH 2 PO 4 1.00 g, NaHCO 3 10.00 g, NaCl 2.00 g and Water distilled 1000.00 mL.
    [0185] • Hemin solution: Dissolve 50 mg of hemin in 1 mL 1 N NaOH; bring it to 100 mL with distilled water. Keep refrigerated.
    [0186] • Vitamin K1 solution: Dissolve 0.1 mL of vitamin K1 in 20 mL of 95% ethanol and filter sterilize. Store refrigerated and protected from light.
    [0188] The ingredients were dissolved (except for cysteine, hemin, and vitamin K1) and autoclaved for 20 minutes at 121 ° C. It was allowed to cool and the cysteine, vitamin K1 and hemin were added later. The pH was adjusted to 7.2 and the medium was placed in an anaerobic chamber to guarantee its anoxic state prior to the inoculation of P. faecium G104.
    [0190] EXAMPLE 2. Selection of P. faecium based on its ability to modulate inflammation in vitro .
    [0191] In vitro tests were performed to comparatively evaluate the immunomodulatory properties of bacteria of human intestinal origin and select the strain capable of inducing the highest anti-inflammatory response in classic monocytes and therefore with potential therapeutic interest in the treatment of inflammation associated with obesity. For this, cell suspensions of different bacterial strains were used as a stimulus in peripheral blood mononuclear cell cultures (PBMCs) and the number of classic monocytes and the levels of the anti-inflammatory cytokine IL-4 were measured by means of proinflammatory cytokine IFNy by cytometry flow.
    [0193] Culture and stimulation of PBMCs.
    [0194] From the whole blood of healthy volunteers, Peripheral Blood Mononuclear Cells (PBMCs) were isolated using a Ficoll gradient (Ficoll Paque-Plus 17 1440-02, Bioscience). After treating them with a solution to lyse the erythrocytes (Lysis Buffer for Red Blood Cells, RBC, Miltenyi Biotec., Spain) they were resuspended in RPMi 1640 medium (Gibco, Barcelona, Spain) supplemented with 10% fetal bovine serum (Gibco , Barcelona, Spain), streptomycin (100 pg / mL, Sigma), penicillin (100 U / mL, Sigma) and L-glutamine (Sigma). To perform the experiments, the PBMCs were incubated at a concentration of 106 per mL in 24-well flat-bottom polystyrene plates (Corning, Madrid, Spain) at 37 ° C, 5% CO2. Live bacteria suspensions at a concentration of 107 cfu / mL were used as a stimulus. As a positive control, purified lipopolysaccharide (LPS) from Salmonella enterica serotype Typhimurium (Sigma Chemical Co, Madrid, Spain) was used at a concentration of 1 pg / mL and samples of untreated PBMCs were used as a negative control. The stimulation time was 24 hours at 37 ° C, at 5% CO2. At the end of this time, the cells were collected and centrifuged, separating the cell pellet from the supernatant. Each type of stimulus was tested in triplicate in 3 independent experiments. The culture supernatants were fractionated and stored in aliquots at -80 ° C.
    [0196] Characterization of the immunomodulatory properties of bacterial strains on PBMCs by flow cytometry.
    [0198] The stimulated PBMCs were analyzed by flow cytometry in order to determine the levels of classic pro-inflammatory monocytes, using the CD14 markers and CD16. In addition, the levels of the proinflammatory cytokine IFNy and the
    [0199] levels of the anti-inflammatory cytokine IL-4 in monocytes. To do this, the cells
    [0200] were permeabilized and fixed (Fixation / Permeabilization Solution Kit, BD-Bioscience)
    [0201] and resuspended with the FACS solution (PBS1X BSA 0.2%). The levels of the
    [0202] Markers were measured using BD LSRFortessa.
    [0204] The comparative evaluation of the different bacterial strains allowed us to conclude that the
    [0205] strain of the invention Phascolarctobacteríum faecium DSM 32890 was the one that induced the
    [0206] more significant immunomodulatory effects, showing a higher production of the
    [0207] anti-inflammatory cytokine IL-4 with respect to pro-inflammatory IFNy (increased IL-4 / IFN y ) and reduction of classical monocytes (CD14 ++ CD16 ") with respect to non-cells
    [0208] treated and treated with LPS, which is an inducer of inflammation associated with the
    [0209] obesity and its complications (Table 1).
    [0211] Table 1: In vitro characterization of the immunomodulatory properties of
    [0212] human intestinal bacteria on PBMCs.
    [0214] Bacterial Strains Classic Monocytes IL-4 / IFNy
    [0215] Untreated 0.71 (0.02) b 1.74 (0.24) d LPS 1.00 (0.05) a 0.80 (0.05) e Alistipes indistinctus 0.69 (0.03) b 3.73 (0.26) b Phascolarctobacterium
    [0216] faecium 0.48 (0.04) c 4.79 (0.08) a Bacteroides dorei 0.70 (0.06) b 3.55 (0.20) b Eubacterium cylindroids 0.75 (0.04) b 1.61 (0.10) d Eubacterium limosum 0.93 (0.03) to 1.39 (0.07) d
    [0218] The results are expressed as the mean and its standard error of the levels.
    [0219] Relatives of classical monocytes and the IL-4 / IFNy ratio measured by flow cytometry.
    [0220] Significant differences (P <0.05) between groups were established by ANOVA
    [0221] of a factor followed by the Tukey post-hoc test. Different letters indicate differences
    [0222] significant between experimental groups (p <0.05).
    [0224] EXAMPLE 3. Effects of P. faecium in an animal model of obesity
    [0226] Development of the animal model of obesity and sampling.
    [0228] C57BL / 6 adult male mice (6-8 weeks, Charles River, Les Oncins, France), maintained under controlled conditions of temperature (23 ° C), relative humidity (40-50%) and 12-hour light / dark cycle, they were fed a hypercaloric diet rich in fat (45% Kcal) and sugar (sucrose); 17 % Kcal) (HFHSD; D12451, Research diet, Brogaarden, Denmark) or with a control diet (CD, 10% Kcal from fat, without sucrose; D12450K, Research diet, Brogaarden, Denmark) for 14 weeks. Daily, the mice fed the HFHSD diet received an oral dose of the bacterial strain object of the invention [(1x107-1x10-8) colony forming units (CFU)] dissolved in 10% skimmed milk. The vehicle (10% skimmed milk) was administered in the same way to both the obese phenotype control group (HFHSD) and the lean phenotype (CD) group (n = 10 mice per group). After 14 weeks, the mice were sacrificed by cervical dislocation to obtain samples, including blood, intestine, liver, brain, inguinal and epididymal white adipose tissue, brown adipose tissue, fecal content and feces.
    [0230] Metabolic phenotype characterization
    [0231] Body weight was monitored weekly. Fasting baseline blood glucose (week 8 and 10) was determined from blood from the saphenous vein using glucose test strips (Contour XT Bayer, Barcelona, Spain) as well as oral glucose tolerance through an oral glucose test (OGTT). , week 10) in which the glycemia was measured at 15, 30, 60 and 120 minutes after having administered an oral glucose overload (2 g / Kg) to mice subjected to 4 hours of fasting.
    [0233] The target bacterium reduced weight gain in the diet-induced obesity model (Figure 1a and 1b), as well as adiposity in obese mice after 14 weeks of treatment (Figure 3c). Furthermore, this bacterium improved both basal glycemia (Figure 2a) and oral glucose tolerance (Figure 2b).
    [0235] Effects on intake
    [0236] The weekly intake recorded in the different cages (5 animals per cage) allowed us to estimate food intake in kilocalories of each of the animals per day (Figure 3a). The observed differences were confirmed by evaluating the individual intake for 24 hours. in week 12 of experiment. The observed result was a reduction in the amount of kilocalories consumed by the group of animals to which the bacteria had been administered, in comparison with the obese group (Figure 3b). The regulation of appetite seems to be one of the mechanisms by which the bacteria under study reduces weight and body fat.
    [0238] Effects on inflammation
    [0239] The effects of P. faecium on intestinal inflammation were analyzed by flow cytometry. To do this, the intestine underwent stages of digestion together with mechanical treatment, which allowed the epithelium to be separated from the lamina propria. The clean and chopped tissue was incubated with shaking for 30 min at 37 ° C with the first pre-digestion solution (5 mM EDTA, 1 mM DTT, 100 pg / ml streptomycin and 100 U / ml penicillin in HBSS [Hank's Balanced Salt Solution ]). This process was repeated twice, filtering the tissue with 100pm filters, thus obtaining the cells of the intestinal epithelium. To obtain the cells of the lamina propria the remaining tissue was treated with the digestion solution (0.5 mg / mL Collagenase D, 50 U / mL DNase I, 3mg / mL Dispase II, 100 pg / ml streptomycin and 100 U / ml penicillin in HBSS) with stirring for 30 min at 37 ° C. The process was repeated twice and filtered using 70pm filters.
    [0241] The cell suspensions obtained were treated with antibodies of different extracellular and intracellular markers. Specifically, innate type 1 lymphoid cells were determined in the epithelium (using the Lineage, T-bet and IFNy markers). The population of macrophages M1 (F4 / 80, CD80 and Inos), M2 (F4 / 80, CD80 and Inos) and Treg (CD3, CD4, CD8, CD25 and Foxp3) were determined in the lamina propria. Cells were diluted in FACS solution (1X PBS with 0.2% BSA) and analyzed on a BD LSRFortessa flow cytometer (Becton Dickinson, NJ, USA).
    [0243] In particular, the levels of innate lymphoid cells (ILC) in the epithelium (Innate Lymphoid Cells) were analyzed. These cells are responsible for protecting epithelial barriers against pathogens and maintaining tissue homeostasis. However, group 1 ILCs (ILC1), can promote inflammation in tissue by IFNy production and are increased in obese mice. The evaluated bacteria was able to reduce the proportion of ILC1, thus reducing the inflammation triggered by these cells (Figure 4a).
    [0245] The bacterial strain also reduced the polarization of macrophages towards a phenotype. M1 (pro-inflammatory) that can be induced by increased IFNy production. On the contrary, it increased M2 levels, inducing an anti-inflammatory response (Figure 4b), normalizing the M1 / M2 ratio (Figure 4c).
    [0247] Furthermore, P. faecium attenuated the effects of obesity by inducing a Th2 / Treg-type response (Figures 4d and 4e). This response was evaluated by measuring the levels of Gata3 as a transcription factor mediating a Th2 response and the levels of regulatory T cells with the CD25 and FoxP3 markers. The increase in Gata3 could also indicate an increase in the proportion of ILC2 cells and a development of ILC3. ILC2s are characterized by producing Th2-type cytokines, with an anti-inflammatory effect in the context of obesity. The Gata3 transcription factor has also been described in a subset of ILC3 associated with the intestinal mucosa that could contribute to its protection.
    [0249] Finally, P. faecium was able to reverse the increase in the proportion of intraepithelial lymphocytes (IEL) induced by the hypercaloric diet, and to normalize the ratio of natural / induced IELs (Figure 5), which would also contribute to restoring homeostasis. immunological and the intestinal barrier avoiding systemic inflammation.
    权利要求:
    Claims (20)
    [1]
    1. A strain of Phascolarctobacteríum faecium with deposit number DSM 32890.
    [2]
    2. A strain derived from the strain according to claim 1.
    [3]
    3. Strain according to claim 1 or 2, wherein said strain is a genetically modified mutant.
    [4]
    4. Strain according to any one of claims 1 to 3, wherein said strain is in the form of viable cells or in the form of non-viable cells.
    [5]
    5. Cellular component, metabolite, secreted molecule or any of its combinations, obtained from the strain according to any one of claims 1 to 4.
    [6]
    6. Composition comprising a strain according to any of claims 1 to 4, or the cellular component, metabolite, secreted molecule or any of its combinations according to claim 5, or any combination thereof.
    [7]
    7. Composition according to claim 6, further comprising at least one bioactive component.
    [8]
    Composition according to claim 6 or 7, further comprising at least one microorganism other than the strain according to any one of claims 1 to 4.
    [9]
    9. Composition according to claim 8, wherein the microorganism is an intestinal bacterium or a lactic acid bacterium.
    [10]
    Composition according to any one of Claims 6 to 9, wherein said composition is a pharmaceutical composition.
    [11]
    11. Composition according to claim 10, wherein the composition additionally comprises at least one vehicle and / or one pharmaceutically acceptable carrier.
    [12]
    12. Composition according to claim 10 or 11, wherein said composition is presented in a form adapted for oral, sublingual, nasal, intracathecal, bronchial, lymphatic, rectal, transdermal, inhaled or parenteral administration.
    [13]
    13. Composition according to any of claims 6 to 9, wherein said composition is a nutritional composition.
    [14]
    14. Composition according to claim 13, wherein said nutritional composition is a food, a supplement, a nutraceutical, a probiotic or a symbiotic.
    [15]
    15. Composition according to claim 14, wherein said food is selected from the list consisting of a dairy product, a vegetable product, a meat product, a snack, chocolate, drink or baby food.
    [16]
    16. Composition according to any one of Claims 6 to 15, wherein said composition has a concentration of the strain of between 103 and 1014 colony forming units (cfu) per gram or milliliter of final composition.
    [17]
    17. Strain according to any of claims 1 to 4, or cellular component, metabolite, secreted molecule or any of its combinations according to claim 5, or the composition according to any one of claims 6 to 16, for use as a medicine.
    [18]
    18. Strain according to any one of claims 1 to 4, a cellular component, metabolite, secreted molecule or any of its combinations according to claim 5, or a composition according to any one of claims 6 to 16, for use in prevention. and / or the treatment of overweight and / or obesity, or diseases associated with it.
    [19]
    19. The strain, cellular component, metabolite, secreted molecule, composition or any of its combinations, according to claim 18, wherein the diseases associated with overweight and / or obesity are selected from the list consisting of cardiovascular diseases, metabolic syndrome, diabetes, hyperglycemia, insulin resistance, cancer, hypertension, dyslipidemia, hypolipidemia, galactosemia, phenylketonuria, sitosterolemia, hyperthyroidism, and hypothyroidism.
    [20]
    20. Non-therapeutic use of a strain according to any one of claims 1 to 4, a cellular component, metabolite, secreted molecule or any of its combinations according to claim 5, or a composition according to any one of claims 13 to 16, for the regulation of appetite.
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    同族专利:
    公开号 | 公开日
    EP3889250A1|2021-10-06|
    ES2763874B2|2020-10-13|
    CN113544255A|2021-10-22|
    CA3121419A1|2020-06-04|
    WO2020109646A1|2020-06-04|
    US20210369793A1|2021-12-02|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    JP2013102714A|2011-11-11|2013-05-30|Yakult Honsha Co Ltd|New use of bacterium belonging to phascolarctobacterium|
    US20130224155A1|2012-02-29|2013-08-29|The General Hospital Corporation D/B/A Massachusetts General Hospital|Compositions of microbiota and methods related thereto|
    WO2018117263A1|2016-12-23|2018-06-28|Keio University|Compositions and methods for the induction of cd8+ t-cells|
    WO2022020852A1|2020-07-22|2022-01-27|The University Of Chicago|Methods and compositions for treating autoimmune and allergic disorders|
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    PCT/ES2019/070821| WO2020109646A1|2018-11-30|2019-12-02|Phascolarctobacterium faecium for use in the prevention and treatment of obesity and its comorbidities|
    CA3121419A| CA3121419A1|2018-11-30|2019-12-02|Phascolarctobacterium faecium for use in the prevention and treatment of obesity and its comorbidities|
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    CN201980079368.2A| CN113544255A|2018-11-30|2019-12-02|Coprolalia for preventing and treating obesity and complications thereof|
    US17/298,377| US20210369793A1|2018-11-30|2019-12-02|Phascolarctobacterium faecium for use in the prevention and treatment of obesity and its comorbidities|
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