ES2752798B2 - Bifidobacterium longum sub strain. infantis and use of it - Google Patents

Description

D E S C R I P T I O N

Bifidobacterium longum sub strain . infantis and use of it

The present invention relates to the Bifidobacterium longum sub strain . infantis CECT 9720, and to compositions that comprise it, as well as the use of said strain for the prevention and / or treatment of diseases that cause intestinal and / or liver inflammation, as well as pathologies derived from them, such as, for example, hepatic cirrhosis. The present invention falls within the field of therapeutic activity of pharmaceutical compositions or preparations, as well as within the field of food.

BACKGROUND OF THE INVENTION

Humans are home to about 10 trillion intestinal bacteria, and their genome has a coding capacity much higher than that of humans (about 100-150 times higher). The gastrointestinal tract is the largest surface area of the body with an epithelial surface area of approximately 400 m2, constantly exposed to these live microorganisms. The existing symbiosis, demonstrated by the lack of pro-inflammatory response against commensal bacteria (Littman DR et al. Role of the commensal microbiota in normal and pathogenic host immune responses. Cell host & microbe 2011; 10 (4): 311-23; Hooper LV et al J. Interactions between the microbiota and the immune system. Science 2012; 336 (6086): 1268-73) implies the presence of clearly defined lines of communication. Recent literature suggests that organisms of the gastrointestinal tract play an indispensable role in the maintenance of host homeostasis (Wu GD et al. Analysis of the human gut microbiome and association with disease. Clinical gastroenterology and hepatology: the official clinical practice journal of the American Gastroenterological Association 2013; 11 (7): 774-7). Alterations in the gut microbiota appear to play a role in the pathogenesis and progression of various diseases, including, for example, liver and gastrointestinal diseases.

More specifically, the gut microbiota has been shown to play a direct role in the progression and development of complications in liver disease (Bajaj. JS et al. Linkage of gut microbiome with cognition in hepatic encephalopathy.

American journal of physiology Gastrointestinal and liver physiology 2012; 302 (1): G168-75; Bajaj JS et al. Colonic mucosal microbiome differs from stool microbiome in cirrhosis and hepatic encephalopathy and is linked to cognition and inflammation. American journal of physiology Gastrointestinal and liver physiology 2012; 303 (6): G675-85; Chen Y. et al. Characterization of fecal microbial communities in patients with liver cirrhosis. Hepatology 2011; 54 (2): 562-72).

Cirrhosis is the common end stage of different liver diseases characterized by a histological distortion of the liver with the presence of regenerative nodules causing portal hypertension (Bosch J. et al Pathophysiology of portal hypertension. Gastroenterology clinics of North America 1992; 21 (1): 1-14). This fact, in turn, alters intestinal motility inducing intestinal bacterial overgrowth (SBI) (Morencos FC et al. Small bowel bacterial overgrowth in patients with alcoholic cirrhosis. Digestive diseases and sciences 1995; 40 (6): 1252-6; Pande C. Small-intestinal bacterial overgrowth in cirrhosis is related to the severity of liver disease. Alimentary pharmacology & therapeutics 2009; 29 (12): 1273-81; Bauer TM et al. Small intestinal bacterial overgrowth in patients with cirrhosis: prevalence and relation with spontaneous bacterial peritonitis. Am J Gastroenterol 2001; 96 (10): 2962 67). Specifically, in patients with cirrhosis there is an overgrowth of potentially pathogenic bacteria (eg Gram-negative species), and a decrease in autochthonous bacterial families. The close relationship between the most frequent complications that arise in patients with cirrhosis and the intestinal microbiota (edesma F. Small bowel bacterial overgrowth in patients with alcoholic cirrhosis. Digestive diseases and sciences 1995; 40 (6): 1252-6; Pande C . et al. Small-intestinal bacterial overgrowth in cirrhosis is related to the severity of liver disease. Alimentary pharmacology & therapeutics 2009; 29 (12): 1273-81; Bauer TM et al. Small intestinal bacterial overgrowth in patients with cirrhosis: prevalence and relation with spontaneous bacterial peritonitis. Am J Gastroenterol 2001; 96 (10): 2962-67; Chang CS et al. Small intestine dysmotility and bacterial overgrowth in cirrhotic patients with spontaneous bacterial peritonitis. Hepatology 1998; 28 (5): 1187- 90) has been studied intensively in recent years and has highlighted the importance of constant communication between the intestine and the liver during the management of patients with cirrhosis (Garcia-Tsao G. et al. Gut microflora in the pathogenesis of the complications of cirrhosis. Best Practice Res Clin Gastroenterol 2004; 18 (2): 353-72). Complications such as hepatic encephalopathy (HE), spontaneous bacterial peritonitis (SBP), and Variceal bleeding in cirrhotic patients is directly caused or aggravated by the translocation of enteric microbiota or its products to extraintestinal territories.

Delving into the above, direct communication between the liver and the intestine through the portal circulation called the "liver-intestine axis", plays an important role in the progression of complications of cirrhosis, with the intestinal microbiota being a key regulator of inflammation and bacterial translocation in this context (Garcia-Tsao G. et al. Gut microflora in the pathogenesis of the complications of cirrhosis. Best Pract Res Clin Gastroenterol 2004; 18 (2): 353-72).

On the other hand, and in relation to intestinal disease, it is also widely known that the intestinal microbiota has a direct role in the progression and development of complications in this pathology. Thus, for example, endotoxins are immunogenic molecules derived from the cell walls of Gram-negative bacteria present in the intestinal microbiota, which can over-activate the innate immune system and break immune homeostasis and intestinal barrier function. (Rodes L. et al. Microencapsulated Bifidobacterium longum subsp. Infantis ATCC 15697 Favorably Modulates Gut Microbiota and Reduces Circulating Endotoxins in F344 Rats. BioMed Research International. Volume 2014, Article ID 602832, 11 pages).

In routine medical practice, as an alternative or complementary to pharmacological treatments, the use of a wide range of probiotic strains has become widespread in recent years, as there is evidence of their contribution to restoring alterations in intestinal permeability , the intestinal microbial composition and the inflammatory response. In this sense, probiotic strains of species belonging to the genera Bifidobacterium and Lactobacillus have been especially used for the treatment and / or prevention of various pathologies so that in a natural way, they restore a situation of intestinal homeostasis, improving the symbiotic interaction between the microbiota and the immune system of patients, while avoiding the non-selective eradication of microbiota associated with the use of antibiotics and the appearance of resistance, the main problems that occur with the use of antibiotics.

Specifically, the use of strains of Bifidobacterium longum subp.

infantis with the treatment of intestinal pathologies, for example:

- Document WO2009134948A1 discloses the treatment of intestinal inflammatory disorders including irritable bowel syndrome, lymphocytic colitis, collagenous colitis, and indeterminate colitis, by administering a composition comprising an anti-inflammatory active agent and a probiotic such as Bifidobacterium infantis diseases, as an adjuvant,

- Document WO2010105207A1 refers to a composition useful to stimulate the growth of beneficial microbiota, including species of the genus Bifidobacterium, in order to regulate bacterial dysbiosis in an animal. The composition comprises galactooligosaccharides and a Bifidobacterium longum subps strain . infantis and as directed may be used to treat, among other conditions, colitis, Crohn's disease, irritable bowel syndrome and inflammatory bowel disease,

- The publication: Elian, SDA et al. "Bifidobacterium longum subsp infantis BB-02 attenuates acute murine experimental model of inflammatory bowel disease" Beneficial Microbes. 2015, 6 (3), 277 - 286 refers to the use of the bacterium Bifidobacterium longum subsp infantis BB-02 as a probiotic that attenuates an acute murine experimental model of inflammatory bowel disease,

-Finally, the publication: Rodes, L. et al. "Microencapsulated Bifidobacterium longum subsp infantis ATCC 15697 Favorably Modulates Gut Microbiota and Reduces Circulating Endotoxins in F344 Rats" BioMed Research International 2014; 2014: 602832., Describes the application of the Bifidobacterium longum subsp infantis strain ATCC 15697 in microencapsulated form, which modulates the intestinal microbiota and reduces circulating endotoxins in a mouse animal model used in aging, oncology and nutrition.

The identification of new probiotic bacterial strains that have the capacity for the treatment and / or prevention of pathologies that present with inflammation, preferably intestinal and liver, as well as derived pathologies such as cirrhosis, and that are preferably effective is considered of interest. simultaneously against more than one of them.

DESCRIPTION OF THE INVENTION

The present invention relates to the strain Bifidobacterium longum subsp. infantis CECT 9720, and to the compositions that comprise it, as well as its use for the prevention and / or treatment of intestinal and / or liver inflammation, and derived pathologies such as, for example, liver cirrhosis. Preferably the strain of the invention exhibits activity simultaneously in more than one of the indicated diseases.

The CECT 9720 strain, which belongs to the species Bifidobacterium longum subsp. Infantis, reduces in vivo peripheral levels of the pro-inflammatory cytokine TNF-a and IL-6 in a model of liver damage and also reduces in vivo peripheral levels of the pro-inflammatory cytokine TNF-a, IL-6 and MCP-1 in a intestinal inflammation model. Furthermore, this strain exerts its effect without inducing a pro-inflammatory response in vitro in Kupffer cells isolated from the liver or in macrophages isolated from the intestinal wall, which is essential in any strain with potential use as a probiotic. All this makes the strain of the present invention a bacterial strain of potential interest for probiotic use by reducing liver and intestinal inflammation.

Therefore, the present invention brings to the state of the art a strain of the species Bifidobacterium longum subsp. infantis for the treatment of intestinal inflammation, liver inflammation and derived pathologies such as, for example, liver cirrhosis.

Then, one aspect of the present invention relates to a strain of Bifidobacterium longum subsp. infantis with deposit number CECT 9720. This strain has been deposited in the Spanish collection of type cultures (CECT) on September 18, 2018 and corresponded to the deposit number CECT 9720. The address of said International Deposit Authority is: Spanish Collection of Type Crops (CECT), Building 3 CUE, University of Valencia Science Park, Professor Agustín Escardino 9, 46980 Paterna (Valencia) Spain.

Within the scope of this document, the reference to the strain of the invention will be made interchangeably as: Bifidobacterium longum subsp. infantis with deposit number CECT 9720, strain CECT 9720, strain Bifidobacterium longum subsp infantis 16p1 o16p1.

The CECT 9720 strain can be in the form of viable cells or in the form of non-viable cells.

The present invention also contemplates a strain derived from the CECT 9720 strain, where said strain maintains or improves the capabilities described throughout the present invention. The derived microorganism can be produced naturally or intentionally, by means of mutagenesis methods known in the state of the art such as, for example, 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 CECT 9720 strain is a genetically modified mutant. The terms mutant strain or derived strain can be used interchangeably.

Another aspect of the present invention refers to a composition comprising the strain of the invention.

In a preferred embodiment, the composition has a strain concentration of between 105 and 1014 colony forming units (cfu) per gram or milliliter of final composition.

The composition can also include at least one bioactive component (active substance, active principle or therapeutic agent).

The term "bioactive component" refers to a compound with biological activity within the scope of the patent that can improve or complement the activity of the CECT 9720 strain, including ingredients or components of food (for example and without limitation: acids polyunsaturated fatty acids, conjugated linoleic acid, prebiotics, fiber, Guar gum, glucomannan, chitosan, copper picolinate, calcium, etc.), other probiotics, plants, extracts or components of plants and drugs.

The composition may be a pharmaceutical composition, in which case it will preferably comprise at least one pharmaceutically acceptable carrier and / or excipient.

The pharmaceutical composition or medicament can be presented in any clinically permitted form of administration and in a therapeutically effective amount. For example, it may be in a form adapted for oral, sublingual, nasal, intracatecal, 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 a tablet, capsule, powder, granule, ointment, solution, suppository, injection, inhalant, gel, microsphere or aerosol. The form adapted for oral administration is selected from the list that includes, but is not limited to, drops, syrup, herbal tea, elixir, suspension, extemporaneous suspension, drinkable vial, tablet, capsule, granulate, seal, pill, tablet, lozenge, troche or lyophilized.

The term "excipient" refers to a substance that helps the absorption of any of the components of the composition of the present invention, stabilizes said components or helps the preparation of the pharmaceutical composition in the sense of giving it consistency or providing flavors that make it more enjoyable. Thus, excipients could have the function of keeping the components together, such as starches, sugars or celluloses, a sweetening function, a coloring function, a protective function of the drug, such as for example to isolate it from air and / or humidity, a function filling of a tablet, capsule or any other form of presentation such as, for example, dibasic calcium phosphate, 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 material that, included in the galenic forms, is added to the active principles or their associations to enable their preparation and stability, modify their organoleptic properties or determine the physicochemical 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 said components.

The "galenic form" or "pharmaceutical form" is the arrangement to which the active principles and excipients are adapted to constitute a medicine. It is defined by the combination of the form in which the pharmaceutical composition is presented by the manufacturer and the way it is administered.

The "vehicle" or carrier is preferably an inert substance. The function of the vehicle is to facilitate the incorporation of other compounds, allow a better dosage and administration or give consistency and shape to the pharmaceutical composition. Therefore, the vehicle is a substance that It is used in the medicine to dilute any of the components of the pharmaceutical composition of the present invention to a certain volume or weight; or that even without diluting said components it is capable of allowing a better dosage and administration or giving consistency and shape to the medicine When the presentation form is liquid, the pharmaceutically acceptable carrier is the diluent.

Furthermore, the excipient and the vehicle must be pharmacologically acceptable, that is, the excipient and the vehicle must be allowed and evaluated so that it does not cause harm to the organisms to which it is administered.

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 prevention and / or treatment, such as is 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; in whatever form; the therapeutically amount Effective 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; the rate of excretion, the combination of drugs; the severity of the particular disorder or pathological condition; and the subject undergoing therapy, but can be determined by a specific cialist in the art according to his own knowledge and that description.

The composition can also be a nutritional composition such as a food, a supplement, a nutraceutical, a probiotic, or a symbiotic.

The term "nutritional composition" of the present invention refers to that food that, regardless of providing nutrients to the subject who takes it, affects beneficially to one or more functions of the body, in a way that provides 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 factor causing a disease. Therefore, the term "nutritional composition" of the present invention can be used synonymously with functional food or food for specific nutritional purposes or medicinal food.

The term "nutraceutical" as used in the present invention refers to substances isolated from a food and used in a dosage form that have a beneficial effect on health.

The term "probiotic" as used in the present invention refers to live microorganisms that, when supplied in adequate amounts, promote health benefits to the host organism.

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.

The term "supplement", synonymous with any of the terms "dietary supplement", "nutritional supplements"; or "food supplement" is a "food ingredient" intended to supplement the diet. Some examples of dietary supplements are, 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 sole component of a meal or of the nutritional diet but as a complement to the diet.

Another aspect of the present invention relates to the use of a Bifidobacterium longum subsp. infantis CECT 9720 for the manufacture of a medicine from a nutritional composition or from a food.

Another aspect of the present invention relates to the strain Bifidobacterium longum subsp. infantis CECT 9720 for use as a medicine.

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 having 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 modify the physiological functions by exerting a pharmacological, immunological or metabolic action, or to establish a medical diagnosis. The "medicine for veterinary use" 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. Also considered "veterinary drugs" are "medicated feed premixes" prepared to be incorporated into a feed.

Another preferred embodiment of the present invention relates to the use of a CECT 9720 strain, or of the composition of the invention, for the manufacture of a medicament for the prevention and / or treatment of inflammatory bowel disease, liver inflammation and / or Derivative pathologies such as liver cirrhosis.

Another preferred embodiment of the present invention relates to the CECT 9720 strain for use in the prevention and / or treatment of inflammatory bowel disease, liver inflammation and / or derived pathologies such as, for example, liver cirrhosis.

Another preferred embodiment of the present invention relates to the CECT 9720 strain for use in the prevention and / or simultaneous treatment of inflammatory bowel disease and liver inflammation.

In a preferred embodiment, the inflammatory bowel disease or intestinal inflammation is selected from the list comprising: Crohn's disease, ulcerative colitis, indeterminate colitis, collagenous colitis, and lymphocytic colitis.

The term "treatment" as understood in the present invention refers to combating the effects caused as a consequence of a disease or pathological condition of interest in a subject (preferably a mammal, and more preferably a human) including:

(i) inhibiting the disease or pathological condition, that is, arresting its development;

(ii) alleviating the disease or pathological condition, that is, causing regression of the disease or pathological condition or its symptomatology;

(iii) stabilize the disease or pathological condition.

The term "prevention" as understood in the present invention is to prevent the occurrence of the disease, that is, to prevent the disease or pathological condition from occurring in a subject (preferably a mammal, and more preferably a human), in particularly, when said subject has a predisposition for the pathological condition.

Within the scope of the invention, preferably the treatment and / or prevention is effective against more than one disease, preferably inflammatory bowel disease and liver cirrhosis.

The term "liver inflammation" or "liver inflammation" refers to an inflammatory response of the liver that may be initiated by physical injury, infection, or local immune response and may include local accumulation of fluid, plasma proteins, and white blood cells, as well as migration and infiltration of neutrophils, lymphocytes, and other cells of the immune system into regions of the damaged liver.

The term "inflammatory bowel disease" or "intestinal inflammation" refers to an inflammatory response of the intestine that can be initiated by physical injury, infection or local immune response and can include local accumulation of fluid, plasma proteins and white blood cells, as well as migration and infiltration of neutrophils, lymphocytes and other cells of the immune system in the regions of the damaged intestine.

The term "cirrhosis of the liver", "cirrhosis of the liver" or simply "cirrhosis" is the end-stage inflammatory liver disease and refers to a condition in which the liver slowly deteriorates and malfunctions as liver tissue is replaced by fibrous scar tissue and regenerative nodules. This produces a partial blockage in the flow of blood through the liver as well as an impairment of the liver's ability to control infections, remove bacteria and toxins from the blood, process nutrients, hormones and drugs, make proteins that regulate blood clotting. blood and produce bile to help absorb fats, including cholesterol, and fat-soluble vitamins. The strain of the present invention is suitable for the treatment of cirrhosis of different causes, including alcohol-related cirrhosis, chronic hepatitis B, C or D, nonalcoholic hepatic steatosis (NASH), autoimmune hepatitis, primary or secondary biliary cirrhosis, sclerosing cholangitis. primary, inherited diseases such as cystic fibrosis, alpha-1 antitrypsin deficiency, hemochromatosis, Wilson's disease, galactosemia, and glycogen storage diseases. The present invention can be used to prevent or treat liver cirrhosis regardless of its etiology.

Throughout the description and claims the word "comprise" 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 partly from the description and partly 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.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Serum levels of cytokines (IL-6, IL-10, MCP-1, TNF-a) in animals with cirrhosis induced by BDL ((common bile duct ligation, English Bile Duct Ligation), distributed by study groups : Untreated (0), Bifidobacterium bifidum MT7 (MT7), Bifidobacterium bifidum G1 (G1), Bifidobacterium longum subsp infantis 16p1 (CECT 9720) according to the example of the invention.

FIG. 2: Secreted levels of cytokines (IL-6, IL-10, MCP-1, TNF-a) in liver macrophage cultures from animals with BDL-induced cirrhosis: Untreated macrophages (MCF), Macrophage stimulated with exposed to lipopolysaccharide (MCF + LPS), Macrophages stimulated with Bifidobacterium bifidum MT7 (MCF + MT7), Macrophages stimulated with Bifidobacterium bifidum G1 (MCF + G1), Macrophages stimulated with Bifidobacterium longum subsp infantis 16p1, (MCF + 16p1) according to the example of the invention.

FIG. 3: Shows the direct comparison of the use of the Bifidobacterium longum subsp infantis 16p1 strain in Kupffer cells (liver macrophages) of animals with BDL-induced cirrhosis, versus lipopolysaccharide (LPS) and unstimulated cells in the production of cytokines (IL -6, IL-10, MCP-1, TNF-a) in the culture supernatants, discarding the MT7 and G1 strains , according to the example of the invention. Untreated Macrophages (MCF), Lipopolysaccharide Stimulated Macrophage (MCF + LPS), and Bifidobacterium longum subsp infantis 16p1 Stimulated Macrophages, (MCF + 16p1)

FIG. 4: Secreted levels of cytokines (IL-6, IL-10, MCP-1, TNF-a) in the intestinal wall macrophage cultures of animals treated with DSS: Untreated macrophages (MCF), Bifidobacterium bifidum MT7 stimulated macrophages (MCF + MT7), Macrophages stimulated with Bifidobacterium bifidum G1 (MCF + G1), Macrophages stimulated with Bifidobacterium longum subsp infantis 16p1, (MCF + 16p1) according to the example of the invention

FIG. 5: Shows the comparison of the effect of stimulation use of intestinal wall macrophages of animals treated with DSS with the strain Bifidobacterium longum subsp infantis 16p1; with lipopolysaccharide (LPS) and without stimulation in the production of cytokines (IL-6, IL-10, MCP-1, TNF-a) in the culture supernatants, discarding the MT7 and G1 strains, according to the example of the invention . Untreated Macrophages (MCF), Lipopolysaccharide Stimulated Macrophage (MCF + LPS), and Bifidobacterium longum subsp infantis 16p1 Stimulated Macrophages, (MCF + 16p1)

FIG. 6: Serum levels of cytokines (IL-6, IL-10, MCP-1, TNF-a) in animals with intestinal inflammation induced by treatment with DSS, distributed by study groups: Untreated (0), Bifidobacterium bifidum MT7 (MT7), Bifidobacterium bifidum G1 (G1), Bifidobacterium longum subsp infantis 16p1, (16p1) according to the example of the invention.

EXAMPLES

The invention will now be illustrated by means of tests carried out by the inventors, which show the effectiveness of the product of the invention.

Example 1: Treatment of mice with cirrhosis

Isolation and identification of the strains:

Strains of the genus Bifidobacterium were isolated from the feces of healthy infants who had not ingested foods containing bifidobacteria for at least the month prior to analysis and who had not undergone antibiotic treatment. The samples were kept at 4 ° C and analyzed within two hours of their collection. Two grams of each were diluted in 10 mM phosphate buffer with a NaCl content with a concentration of 130 mM (PBS) and homogenized in a Lab-Blender 400 digester (Seward Medical, London, United Kingdom), for 3 min and then diluted in peptone water. 0.1 ml aliquots of various decimal dilutions were inoculated on MRS agar (from Man Rogosa and Sharpe; Scharlau, Barcelona) containing 0.05% cysteine (Sigma, St. Louis, MO; MRS-C) , and 80 ^ g / ml of mupirocin. After an incubation of 48 h at 37 ° C under anaerobic conditions (AnaeroGen, Oxoid, United Kingdom), isolated colonies were selected and their identity was confirmed by a study of their morphology under Gram stain. The identity of the isolates was confirmed by genus-specific PCR, according to the methodology described by Kaufman et al. (1997, Identification and quantification of Bifidobacterium species isolated from food with genus-specific 16S rRNA-targeted probes by colony hybridization and PCR. Appl. Environ. Microbiol. 63: 1268-1273), using the primers (LM26 and LM3) that amplify a 1.35 kb fragment of the 16S ribosomal RNA gene. Its identification at the species level was carried out by sequencing the 16S rRNA gene (1.26 Kb) using the primers 27f (5'-AGAGTTTGATCCTGGCTCAG-3 '(SEQ ID NO: 1) and 1401r (5'-CGGTGTGTACAAGACCC-3' (SEQ ID NO: 2) by Sanger technology on an ABI 3730XL sequencer By comparing the sequences obtained with those of the NCBI database and the BLASTn algorithm, the identification of the isolated strains and, in particular, of the target strains was obtained. of the present patent, obtaining a degree of identity of 100% with Bifidobacterium longum subsp infantis.

Sequence of the 16S rRNA of the Bifidobacterium longum subsp infantis CECT9720 (SEQ ID NO: 3):

TAGCGACTCCGCCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGACCGGTTTT ^{CAGGGATCCGCTCCGCGTCGCCGCGTCGCATCCCGTTGTACCGGCCATTGTAGCATGCGT} GAAGCCCTGGACGTAAGGGGCATGATGATCTGACGTCATCCCCACCTTCCTCCGAGTTAA CCCCGGCGGTCCCCCGTGAGTTCCCGGCATAATCCGCTGGCAACACGGGGCGAGGGTTGC GCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCAC CTGTGAACCCGCCCCGAAGGGAAGCCGTATCTCTACGACCGTCGGGAACATGTCAAGCCC ^{AGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCGCATGCTCCGCCGCTTGTGCGGGCCC} CCGTCAATTTCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGATGCTTAACGC GTTAGCTCCGACACGGAACCCGTGGAACGGGCCCCACATCCAGCATCCACCGTTTACGGC GTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGT AACGGCCCAGAGACCTGCCTTCGCCATTGGTGTTCTTCCCGATATCTACACATTCCACCG ^{TTACACCGGGAATTCCAGTCTCCCCTACCGCACTCAAGCCCGCCCGTACCCGGCGCGGAT} CCACCGTTAAGCGATGGACTTTCACACCGGACGCGACGAACCGCCTACGAGCCCTTTACG CCCAATAATTCCGGATAACGCTTGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTT AGCCGGTGCTTATTCAACGGGTAAACTCACTCACGCTTGCTCCCCGATAAAAGAGGTTTA CAACCCGAAGGCCTCCATCCCTCACGCGGCGTCGCTGCATCAGGCTTGCGCCCATTGTGC ^{AATATTCCCCACTGCTGCCTCCCG TAGGAGTCTGGGCCGTATCTCAGTCCCAATGTGGCC} GGTCGCCCTCTCAGGCCGGCTACCCGTCGAAGCCACGGTGGGCCGTTACCCCGCCGTCAA GCTGATAGGACGCGACCCCATCCCATACCGCGAAAGCTTTCCCAGCAAACCATGCGGAAA GTTGGAGCATCCGGCATTACCACCCGTTTCCAGGAGCTATTCCGGTGTATGGGGCAGGTC GGTCACGCATTACTCACCCGTTCGCCACTCTCACCACCAAGCAAAGCC

Animals and Methods:

Study design:

Male Sprague-Dawley rats (Harían, Barcelona, Spain) weighing 100-150 g were included in this study. Rats were individually housed at a constant room temperature of 21 ° C and exposed to a 12:12 light / dark cycle. The animals were fed standard food throughout the study protocol. The protocol spanned a total of 6 weeks. After one week of quarantine, the animals were subjected to the induction of experimental cirrhosis by double ligation of the common bile duct (Bile Duct Ligation, BDL). Briefly, the rats were anesthetized with ketamine and xylazine. Following a midline laparotomy, the common bile duct was ligated at two different levels with 5-0 silk sutures and the duct was sectioned between the ligatures. The abdominal wall was closed with 5-0 silk sutures. A subset of animals acted as controls and were sham operated.

a) In vivo study :

One week prior to laparotomy, the animals were randomized into 4 groups:

- Without treating

- Treated with Bifidobacterium bifidum MT7

- Treated with Bifidobacterium bifidum G1

- Treated with Bifidobacterium longum subsp infantis 16p1

Each animal included in the different groups received daily a set amount (109 CFU) of the strains included in the study. The administration was carried out by intragastric catheterization and lasted for 7 days.

After finishing the treatment, all the animals were sacrificed and blood samples were collected from the lower cava in heparin tubes. The blood was centrifuged (10 min at 3500 FCR (relative centrifugal force)) to obtain the serum, which was aliquoted and stored at -20 ° C until use.

b) In vitro study:

Animals subjected to the induction of experimental cirrhosis by means of double ligation of the common bile duct (Bile Duct Ligation, BDL) were anesthetized with a mixture of ketamine (75mg / kg) and xylazine (10mg / kg) for the development of laparotomies. Once the liver was located, it was perfused in situ with 50 mL of perfusion buffer (HBSS 10% FBS, 1mg / mL of collagenase I, 0.2mg / mL of collagenase IV, 20 ^ g / mL of DNase I and 5 mM MgCh) previously tempered to 37 ° C. The liver was then removed and placed in a tube with RPMI 1640 at 10% FBS, cut into small pieces and centrifuged at 1300 rpm for 10 min at 4 ° C. The supernatant was carefully removed, the tissue was resuspended in 50 mL of digestion buffer (RPMI 1640, 1mg / mL of collagenase I, 0.2 mg / mL of collagenase IV, 20 ^ g / mL of DNase I and 5 mM MgCh) and incubated for 1 h at 37 ° C under shaking (400 rpms).

The mixture resulting from the digestion was passed through a column of sieves after being completely mechanically disintegrated, washing everything with RPMI 1640 to recover as many cells as possible. This was followed by a very gentle centrifugation (3 min at 30 RCF (relative centrifugal force) and 4 oC) to remove the hepatocytes. The pellet was discarded and the collected supernatant was centrifuged for 10 min at 1300 rpms and 4 ° C. The resulting pellet was separated using a Percoll gradient, which was centrifuged for 10 min at 1500 rpms and without brakes. After collecting the cells from the percoll, they were resuspended in Red Blood Cell Lysing Buffer (Sigma Aldrich) and left to lyse for 10 min at 4 ° C to eliminate the rest of erythrocytes remaining. After the lysing time elapsed, the cells were washed with RPMI 1640 and the total cells were counted.

The isolation of the Kupffer cells was carried out by magnetic separation, according to their expression of the CD68 monocyte differentiation marker. First, a labeling (20min at 4oC) with CD68 antibody bound to PE (Miltenyi Biotec) was performed using 5 ^ L per million cells, followed by a 5min wash at 300gx to eliminate the rest of the antibody not bound to the cells. . A second labeling was then carried out (15 min at 4oC) with anti-PE beads (Miltenyi Biotec) at a rate of 2 ^ L per million cells. After the subsequent washing to eliminate the excess magnetic beads, a positive selection of the sample was carried out, passing the suspension of labeled cells through an LS column (Miltenyi Biotec) to obtain a pure population of Kupffer cells. The cells obtained were resuspended in RPMI 1640 culture medium (Gibco BRL, Life Technologies) supplemented with 10% FBS fetal bovine serum. The viability of the cells was verified by means of a "trypan blue" stain and they were counted. Viable cells were plated at a rate of (106 cells / mL) in 5 wells under the following conditions:

- Not stimulated

- Stimulated with LPS

- In co-culture with Bifidobacterium bifidum MT7 (5x105 CFUs / well)

- In co-culture with Bifidobacterium bifidum G1 (5x105 CFUs / well)

- In co-culture with Bifidobacterium longum subsp infantis 16p1 (5x105 CFUs / well)

After 48 hours of culture, the supernatants were collected and stored at -20 ° C for the subsequent analysis of the inflammatory response. The study was approved by the Animal Research Committee of the Miguel Hernández University (Alicante, Spain) with the code HA-RFG-001-15.

Culture conditions of the strains.

The strains were grown in MRS broth (Scharlau, Barcelona, Spain) supplemented with 0.05% (p / v) cysteine (MRS-C Sigma, St. Louis, MO, USA) and incubated at 37 ° C for 22 h (in stationary growth phase) under anaerobic conditions (AnaeroGen, Oxoid, Basingstoke, UK). Cells were collected by centrifugation (6,000 g for 15 min), washed twice in phosphate-buffered saline (PBS, 130 mM sodium chloride, 10 mM sodium phosphate, pH 7.4) and resuspended in 10% skim milk for oral administration to rats. Aliquots of these suspensions were frozen in liquid nitrogen and stored at -80 ° C until use. The number of live cells was determined using the colony formation unit (CFU) that has MRS-C agar (Biomerieux) after an incubation of 48 h at 37 ° C. For the tested strains, more than 90% of the cells they were alive when thawed and no significant differences were found during storage time (2 months). A fresh aliquot was thawed for each new experiment to avoid variability in culture viability.

ELISAs

The inflammatory responses in serum and culture supernatants were carried out using ELISA-type kits according to the manufacturer's instructions (R&D Systems, Minneapolis, MN). All samples were measured in triplicate in a Sunrise Microplate Reader (Tecan) plate reader and standard curves were generated for each of the plates, establishing in turn a standard zero value that was subtracted from the results of the standards as well as the samples to obtain corrected values.

Statistic analysis

The U-Mann-Whitney test was used for paired comparisons between treatment groups, with the Bonferroni correction for multiple comparisons. The level of significance was 0.05. Statistical analyzes were performed using the SPSS program (version 22.0).

Results:

A total of 28 animals were initially included in the study. Mortality in the BDL group was 23.1% (6 of the 24 animals initially operated on). None of the control rats (n = 4) died during the study protocol. Rats undergoing BDL surgery were randomized to study in vivo (n = 12) or in vitro (n = 6). Within the in vivo study , they were also randomized to belong to any of the treatment groups with the different strains of bifidobacteria (n = 4 / group) the week prior to laparotomy. The expression of soluble inflammatory mediators such as IL-6, IL-10, MCP-1 and the TNF-a in serum and supernatant of the cultures performed.

a) In vivo :

The results of the measurements made in serum (Figure 1) reflected a clear decrease in the pro-inflammatory response. It should be noted that the levels of TNF-a were ten times lower in the group of animals treated orally with Bifidobacterium longum subsp infantis 16p1 compared to the untreated animals. Likewise, a significant decrease was observed in both IL-6 and MCP-1 levels.

On the contrary, the levels of IL-10, a cytokine that regulates the immune response and an inhibitor of pro-inflammatory molecules (among them TNF-a) remained high with Bifidobacterium longum subsp infantis 16p1, as occurred in the basal situation.

b) In vitro:

Figure 2 shows the in vitro production of cytokines measured in the culture supernatant. When evaluating the direct response of the Kupffer cells against each of the studied strains, we observed that the expression of the mediators MCP-1, and IL-6 was increased in all cases with respect to the cells that were not stimulated. however, this increase was only similar to that of LPS-stimulated macrophages in cultures treated with strains MT7 and G1. Cells treated with Bifidobacterium longum subsp infantis 16p1 barely increased IL-6 production, unlike what happened with LPS or the rest of the strains studied. Similarly, in the presence of this strain, the levels of TNF-a were not increased with respect to basal conditions. In addition, to these results was added a decrease in the production of the chemotactic protein MCP-1, involved in the recruitment of monocytes and basophils towards areas of inflammation.

Figure 3 shows the direct comparison of the use of the Bifidobacterium longum subsp infantis 16p1 strain in Kupffer cells against LPS and unstimulated cells in the production of cytokines in the culture supernatants, discarding the MT7 and G1 strains. As can be clearly seen, the use of the strain of the invention maintained cytokine levels at concentrations similar to those of non-cells. stimulated. That is, the Bifidobacterium longum subsp infantis 16p1 strain does not induce the secretion of inflammatory mediators in Kupffer cells.

Conclusion:

With these results, it can be concluded that the Bifidobacterium longum subsp infantis 16p1 strain reduces in vivo peripheral levels of the pro-inflammatory cytokine TNF-a and IL-6 in a model of liver damage. Furthermore, this strain exerts its effect without inducing a pro-inflammatory response in vitro in Kupffer cells isolated from the liver, which is essential in any strain with potential use as a probiotic. All this makes the Bifidobacterium longum subsp infantis 16p1 strain a bacterial strain of potential interest for probiotic use by reducing the inflammation induced in an experimental model of liver damage by cholestasis.

Example 2: Treatment of mice with intestinal inflammation Animals and Methods:

Study design:

Male Sprague-Dawley rats (Harlan, Barcelona, Spain) weighing 100-150 g were included. Rats were individually housed at a constant room temperature of 21 ° C and exposed to a 12:12 light / dark cycle. The animals were fed standard food throughout the study protocol. The protocol spanned a total of 6 weeks. After a week of quarantine, the animals were subjected to inflammatory bowel disease by treatment with 3% DSS in the drinking water. Laparotomy and sampling was performed under Isofluorane anesthesia one week after the start of treatment.

A) In vitro study : The intestine was isolated and cut into 1.5cm pieces, which were placed in a 50mL tube preheated with HBSS / FBS and 2mM EDTA, and shaken twice at 250rpm for 20 minutes at 37 ° C. The intestinal pieces were then transferred to a solution with collagenase (1.5mg / mL of collagenase VIII in HBSS / FBS with 40 µg / mL of DNAse I), and they were shaken at 200rpm for 15 minutes at 37 ° C. The tissue remains were filtered (100 µm) into another 50mL tube. The resulting solution was centrifuged again at 1500rpm for 5 minutes at 4 ° C until a cell pellet was obtained and the supernatant was eliminated.

The isolation of intestinal macrophages was carried out by magnetic separation, according to their expression of the CD45 macrophage differentiation marker, F4 / 80 + and CD11c.

The extracted macrophages were cultured for 48 hours alone (106 cells / mL RPMI), with LPS, or with each of the three strains to be analyzed:

+ Bifidobacterium bifidum MT7 (5x105 CFUs / well),

+ Bifidobacterium bifidum G1 (5105 CFUs / well)

+ Bifidobacterium longum subsp infantis 16p1 (5105 CFUs / well).

After 48 hours of culture, the supernatants were stored at -20 ° C until the inflammatory response was evaluated using ELISA kits (IL-6, IL-10, MCP-1,

TNF-a).

B) In vivo study : One week prior to laparotomy, coinciding with the treatment with DSS, the animals were distributed into groups and randomly received one of the strains included in the study (109 CFU / oral / day / 7 days) .

The animals were divided into 4 groups: 1) untreated, 2) B. bifidum MT7, 3) B. bifidum G1 and 4) B. longum subsp infantis16p1. All the animals were sacrificed at the end of the treatment and

Serum samples that were stored at -20 ° C until measurements were made to evaluate the inflammatory response using ELISA Kits (IL-6, IL-10, MCP-1, TNF-a).

The cultivation conditions of the strains, the performance of the different ELISA kits and the statistical analysis were carried out in the same way as in example 1.

Results:

A total of 22 animals were initially included in the study. None of the DSS-treated rats (n = 18) nor any of the control rats (n = 4) died during the study protocol. The rats were randomized to study in vivo (n = 12) or in vitro (n = 6). Within the in vivo study , they were also randomized to belong to any of the treatment groups with the different strains of bifidobacteria (n = 4 / group) the week prior to laparotomy. The expression of soluble inflammatory mediators such as IL-6, IL-10, MCP-1 and TNF-a in the serum and the supernatant of the cultures performed was quantified.

a) in vitro:

Figure 4 shows the in vitro production of cytokines measured in the culture supernatant. When evaluating the direct response of intestinal macrophages against each of the strains studied, we observed that the expression of the mediators MCP-1, IL-6 and TNF-a was increased in all cases with respect to cells that were not However, this increase was only similar to that of macrophages stimulated with LPS in the cultures treated with the MT7 and G1 strains. Cells treated with Bifidobacterium longum subsp infantis 16p1 barely increased IL-6 production, unlike what happened with LPS or the rest of the strains studied. Similarly, in the presence of this strain, the levels of TNF-a were not increased with respect to basal conditions.

Figure 5 shows the comparison of the direct effect of the Bifidobacterium longum subsp infantis 16p1 strain on intestinal macrophages compared to that of LPS and unstimulated cells in the production of cytokines in the culture supernatants, discarding the MT7 and G1 strains. The use of the strain of the invention maintained the levels of pro-inflammatory cytokines at concentrations similar to those of the unstimulated cells. In other words, the Bifidobacterium longum subsp infantis 16p1 strain does not induce the secretion of inflammatory mediators in intestinal macrophages.

b) Live :

The results of the measurements made in serum (Figure 6) reflected a clear decrease in the pro-inflammatory response. Both the levels of TNF-a, as well as the levels of Il-6 and MCP-1 were lower in the group of animals treated orally with Bifidobacterium longum subsp infantis 16p1 with respect to the untreated animals.

On the contrary, IL-10 levels remained high with Bifidobacterium longum subsp infantis 16p1, as occurred in the basal situation.

Conclusion:

With these results, it can be concluded that the Bifidobacterium longum subsp infantis 16p1 strain reduces the peripheral levels in vivo of the pro-inflammatory cytokine TNF-a, IL-6 and MCP-1 in a model of intestinal inflammation. Furthermore, this strain exerts its effect without inducing a pro-inflammatory response in vitro in macrophages isolated from the intestinal wall, which is essential in any strain with potential use as a probiotic. All this makes the Bifidobacterium longum subsp infantis 16p1 strain a bacterial strain of potential interest for probiotic use by reducing the inflammation induced in an experimental model of intestinal inflammation.

Claims (18)

1. Strain of Bifidobacterium longum sub. infantis with deposit number CECT 9720. 2. A strain according to claim 1, wherein said strain is in the form of viable cells or in the form of non-viable cells. 3. Composition comprising the strain according to any of claims 1 to 2. Composition according to claim 3, which additionally comprises at least one bioactive component. Composition according to any one of claims 3 to 4, wherein said composition is a pharmaceutical composition. Composition according to claim 5, additionally comprising at least one pharmaceutically acceptable carrier and / or excipient. Composition according to any one of claims 5 to 6, wherein said composition is presented in a form adapted for oral, sublingual, nasal, intracathecal, bronchial, lymphatic, rectal, transdermal, inhaled or parenteral administration. Composition according to claim 7, wherein said composition is presented in a form adapted for oral administration. Composition according to any of claims 3 to 4, wherein said composition is a nutritional composition. Composition according to claim 9, wherein the composition is selected from a food, a supplement, a nutraceutical, a probiotic or a symbiotic. Strain according to any one of claims 1 to 2, or of the composition according to any of claims 3 to 4, for use as a medicine. 12. Strain or composition for use according to claim 11 for the prevention and / or treatment of liver inflammation. 13. Strain or composition for use according to claim 11 for prevention and / or treatment of liver cirrhosis. 14. Strain or composition for use according to claim 11 for the prevention and / or treatment of inflammatory bowel disease. 15. Strain or composition for use according to claim 11 for the prevention and / or simultaneous treatment of inflammatory bowel disease and liver inflammation. 16. Strain or composition for use according to claim 11 for the prevention and / or simultaneous treatment of inflammatory bowel disease and liver cirrhosis. 17. Strain or composition for use according to any of claims 14 to 16 wherein the inflammatory bowel disease is selected from the list comprising: Crohn's disease, ulcerative colitis, indeterminate colitis, collagenous colitis, lymphocytic colitis. 18. Use of the strain according to any one of claims 1 to 2, or of the composition according to any of claims 3 to 4 for the preparation of a food.