Serpin production

Abstract

To provide a medium for increasing serpin production in a bacterium expressing serpin, the bacterium, and a composition comprising the bacterium.SOLUTION: The present invention provides a medium comprising a fructose disaccharide at concentration of 0.5 to 1 wt.% or a medium comprising a fructooligosaccharide (FOS) at concentration of 0.5 to 1 wt.% for increasing protein production in Bifidobacterium longum subsp. longum.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to bacteria expressing serpins, methods for increasing serpin production in bacteria, and their use.

[0002] [Background Art] Gluten-related disorders include all diseases induced by gluten. Among these diseases, celiac disease and non-celiac gluten sensitivity are mentioned among several pathophysiological conditions. Currently, the incidence of various gluten-related disorders is increasing worldwide, especially with respect to celiac disease and non-celiac gluten sensitivity. Both diseases are induced by gluten intake. Both innate immunity and adaptive immunity are involved in celiac disease, and innate immunity is involved in non-celiac gluten sensitivity.

[0003] A lifelong gluten-free diet is a typical treatment for patients with celiac disease and non-celiac gluten sensitivity, but it may have certain limitations for extra-intestinal symptoms of the disease (Sedghizadeh et al., 2002, Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology, 94(4), 474-478). It has been shown to be very difficult to follow a strict gluten-free diet because it is difficult to avoid secondary contamination at low levels, which may occur throughout the food production chain from grain growth to manufacturing and processing (Mitchison et al., 1991, Gut, 32(3), 260-265). Furthermore, it has been reported that even under a strict gluten-free diet, it is possible to inadvertently ingest up to 3 g of gluten per day (Aziz et al., 2014, The American journal of gastroenterology, 109(9), 1498).

[0004] Celiac disease is widespread, particularly in the United States and Europe, with approximately 1% of the population testing positive for antibodies (Dube et al., 2005, Gastroenterology, 128(4), S57 - S67). Celiac disease is a complex disorder resulting from the intricate interplay between various immunological, genetic, and environmental factors (Alaedini & Green, 2005). This disease is induced by the digestion of wheat gluten and other related cereal proteins such as rye and barley proteins. Symptoms associated with celiac disease include growth retardation, irritability, and delayed puberty in children, as well as numerous gastrointestinal symptoms such as discomfort, diarrhea, occult stool, steatorrhea, and flatulence (Dube et al., 2005; Sedghizadeh et al., 2002).

[0005] Non - celiac gluten sensitivity (also known as non - celiac wheat sensitivity) is a newly emerging condition. This condition is defined as a clinical entity that causes intestinal and / or extra - intestinal symptoms induced by gluten intake and can potentially be improved by removing gluten - containing foods from the diet (Lundin & Alaedini, 2012). In addition to gliadin (the major cytotoxic antigen of gluten), other proteins / peptides present in gluten and gluten - containing cereals (wheat, rye, barley, and their derivatives) may be involved in the appearance of symptoms. Non - celiac gluten sensitivity is the most common syndrome among gluten - related disorders, showing a prevalence of 0.5 - 13% (mean 5%) in the general population (Catassi et al., 2013, Nutrients, 5(10), 3839 - 3853).

[0006] Serine protease inhibitors (serpins) are a superfamily of proteins found in eukaryotes (Gettins, 2002 Chemical reviews, 102(12), 4751 - 4804) and prokaryotes (Kantyka et al., Biochimie, 92(11), 1644 - 1656).

[0007] In recent years, human serine protease inhibitors have been shown to be strongly involved in gluten-related disorders. Elafin is a human serine protease inhibitor that exhibits potent inhibitory activity against various forms of elastase and proteinase (Ying & Simon, 1993, Biochemistry, 32(7), 1866-1874). Elafin is expressed throughout the gastrointestinal epithelium, and its expression and induction are decreased in patients with inflammatory bowel disease and celiac disease (Baranger, Zani, Labas, Dallet-Choisy, & Moreau, 2011; Motta et al., 2012). In recent years, elafin has been identified as a substrate for the transglutaminase 2 (TG2) cross-linking activity (Baranger et al., 2011, PloS one, 6(6), e20976; Motta et al., Science translational medicine, 4(158), 158ra144-158ra144). In vitro data show that the addition of elafin moderately inhibits transglutaminase 2 (TG2), and thus inhibits the deamidation of the digestion-resistant 33-mer gliadin peptide, which is considered to be one of the possible triggers of the adaptive immune response in celiac disease (McCarville et al. 2015, Current opinion in pharmacology, 25, 7-12).

[0008] Delivery of elafin produced by recombinant Lactococcus lactis has been shown to reduce gluten-induced pathologies and normalize intestinal inflammation in a mouse model of gluten hypersensitivity (Galipeau et al., 2014, The American journal of gastroenterology, 109(5), 748-756). However, this proposed therapy is based on genetically modified microorganisms (GMOs), and due to the very low acceptance of GMOs by consumers, it is not suitable for food applications.

[0009] More recently, prokaryotic serpins have been reported. In silico analysis has revealed that genes encoding serpin-like proteins are present in various Bifidobacterium species, particularly in bacteria of the subspecies longum of the Bifidobacterium longum species. The protein encoded by B. longum subsp. longum (designated B. longum) NCC 2705 exhibited protease inhibitory activity similar to that of human serpins (Ivanov et al 2006, Journal of Biological Chemistry, 281(25), 17246-17252).

[0010] B. longum NCC 2705 was deposited with the Institut Pasteur under the Budapest Treaty on January 29, 2001, and was assigned the deposit number CNCM I-2618.

[0011] More recently, it has been shown that B. longum NCC 2705 (CNCM I-2618) can improve gluten-induced pathophysiology through serpin production in a mouse model of gluten sensitivity, indicating its potential as a solution for gluten-related disorders (McCarville et al., 2017, Appl. Envoron. Microbiol. Vol. 83, no. 19, e01323-17).

[0012] [Summary of the Invention] The inventors have surprisingly found that the addition of fructooligosaccharides and fructooligosaccharides (FOS) to the growth medium of bacteria of the subspecies longum of the Bifidobacterium longum species can increase the production of serpins.

[0013] Accordingly, in a first aspect of the present invention, there is provided the use of fructooligosaccharides or fructooligosaccharides (FOS), or a combination thereof, for increasing serpin production in Bifidobacterium longum subsp. longum.

[0014] In another aspect of the present invention, there is provided a method for increasing serpin production in bacteria of the subspecies longum of the species Bifidobacterium longum, the method comprising the step of growing Bifidobacterium longum subspecies longum in a culture medium, characterized in that the culture medium contains fructose disaccharide or fructooligosaccharide (FOS), or a combination thereof.

[0015] According to another aspect of the present invention, there is provided a bacterium of the subspecies longum of the species Bifidobacterium longum prepared by a method of growing Bifidobacterium longum subspecies longum in a culture medium, characterized in that the culture medium contains fructose disaccharide or FOS, or a combination thereof.

[0016] The Bifidobacterium longum subspecies longum prepared according to the present invention has an increased serpin protein level compared to the same Bifidobacterium longum subspecies longum strain grown in the absence of fructose disaccharide or FOS, or a combination thereof.

[0017] According to the present invention, the Bifidobacterium longum subspecies longum may be cultured in a medium containing fructose disaccharide or FOS, or a combination thereof, for example, at a concentration of 0.02 to 5% by weight, preferably 0.05 to 2% by weight.

[0018] For example, B. longum strain CNCM I-2618 may be cultured in a medium containing fructose disaccharide or FOS, or a combination thereof, at a concentration of 0.02 to 5% by weight, 0.05 to 2% by weight, 0.1 to 1.5% by weight, or about 1% by weight.

[0019] In some preferred embodiments, the fructose disaccharide is selected from sucrose, lactulose, or a combination thereof. In some preferred embodiments, sucrose is used.

[0020] In some preferred embodiments, the FOS is short-chain FOS having an average DP of 2 to 10.

[0021] According to another aspect of the present invention, there is provided a composition comprising Bifidobacterium longum subsp. longum prepared by the method described herein.

[0022] In one embodiment, the composition is a food, a medical food, enteral nutrition, or a nutritional supplement.

[0023] In one embodiment, the food is selected from milk, yogurt, kefir, cheese, fermented milk, milk-based fermented products, rice-based products, milk-based powders, infant formulas, and pet foods.

[0024] In one embodiment, the composition is a pharmaceutical composition, which comprises one or more pharmaceutically acceptable carriers, diluents, and / or additives.

[0025] According to another aspect of the present invention, there is provided Bifidobacterium longum subsp. longum prepared by the method described herein, or a composition comprising the Bifidobacterium longum subsp. longum, for use in the treatment or prevention of a condition associated with gluten hypersensitivity or a condition accompanied by a decrease in the activity of a serine protease inhibitor.

[0026] According to another aspect of the present invention, there is provided Bifidobacterium longum subsp. longum prepared by the method described herein, or a composition comprising the Bifidobacterium longum subsp. longum, for use in the treatment or prevention of gluten-related disorders.

[0027] According to one aspect of the present invention, there is provided Bifidobacterium longum subsp. longum prepared by the method described herein, or a composition comprising the Bifidobacterium longum subsp. longum, for use in the treatment or prevention of celiac disease, non-celiac gluten hypersensitivity, gluten ataxia, dermatitis herpetiformis, or wheat allergy.

[0028] According to another aspect of the present invention, there is provided Bifidobacterium longum subsp. longum prepared by the method described herein, or a composition comprising said Bifidobacterium longum subsp. longum, for use in the treatment or prevention of inflammatory bowel disease.

[0029] The Bifidobacterium longum subsp. longum may be any Bifidobacterium longum subsp. longum strain. In some preferred embodiments, the Bifidobacterium longum subsp. longum strain is Bifidobacterium longum subsp. longum strain CNCM I-2169, Bifidobacterium longum subsp. longum strain CNCM I-2171, Bifidobacterium longum subsp. longum strain ATCC BAA-999, Bifidobacterium longum subsp. longum strain ATCC 15708, Bifidobacterium longum subsp. longum strain DSM 20097, Bifidobacterium longum subsp. longum strain NCIMB 8809, Bifidobacterium longum subsp. longum strain CNCM I-2618 (NCC 2705), Bifidobacterium longum subsp. longum strain CNCM I-2170, Bifidobacterium longum subsp. longum strain ATCC 15707 (T), or a combination thereof, particularly selected from B. longum CNCM I-2618 (NCC 2705).

[0030] In some embodiments, the fructooligosaccharide is selected from sucrose, lactulose, or a combination thereof. In some preferred embodiments, sucrose is used.

[0031] In some embodiments, the FOS has a DP in the range of 2 to 10, preferably 2 to 8.

[0032] It will also be understood that when the fructooligosaccharide or FOS, or a combination thereof, is administered in combination with Bifidobacterium longum subsp. longum, the fructooligosaccharide and / or FOS can also increase serpinin production in Bifidobacterium longum subsp. longum in vivo.

[0033] Accordingly, according to another aspect of the invention, there is also provided a combination of (i) Bifidobacterium longum subsp. longum and (ii) fructooligosaccharide or FOS, or a combination thereof.

[0034] According to another aspect of the invention, there is also provided a combination of (i) Bifidobacterium longum subsp. longum and (ii) fructooligosaccharide or FOS, or a combination thereof, for use in the treatment or prevention of a condition associated with gluten hypersensitivity or a condition associated with a decrease in the level of a serine protease inhibitor.

[0035] In one embodiment, the combination is a combination of B. longum strain CNCM I-2618 and sucrose.

[0036] In another embodiment, the combination is a combination of B. longum strain CNCM I-2618 and FOS, preferably FOS having a DP in the range of 2 to 10, preferably 2 to 8.

[0037] According to another aspect of the invention, there is also provided Bifidobacterium longum subsp. longum for use in the treatment or prevention of a condition associated with gluten hypersensitivity or a condition associated with a decrease in the level of a serine protease inhibitor, wherein the Bifidobacterium longum subsp. longum is administered in combination with fructooligosaccharide or FOS, or a combination thereof.

[0038] According to another aspect of the invention, there is also provided fructooligosaccharide or FOS, or a combination thereof, for use in the treatment or prevention of a condition associated with gluten hypersensitivity or a condition associated with a decrease in the level of a serine protease inhibitor, wherein the fructooligosaccharide or FOS, or a combination thereof, is administered in combination with Bifidobacterium longum subsp. longum.

[0039] In some embodiments, Bifidobacterium longum subsp. longum may be selected from Bifidobacterium longum subsp. longum strain CNCM I-2169, Bifidobacterium longum subsp. longum strain CNCM I-2171, Bifidobacterium longum subsp. longum strain ATCC BAA-999, Bifidobacterium longum subsp. longum strain ATCC 15708, Bifidobacterium longum subsp. longum strain DSM 20097, Bifidobacterium longum subsp. longum strain NCIMB 8809, Bifidobacterium longum subsp. longum strain CNCM I-2618 (NCC 2705), Bifidobacterium longum subsp. longum strain CNCM I-2170, Bifidobacterium longum subsp. longum strain ATCC 15707 (T), or combinations thereof.

[0040] In some preferred embodiments, Bifidobacterium longum subsp. longum may be selected from Bifidobacterium longum subsp. longum strain CNCM I-2169, Bifidobacterium longum subsp. longum strain CNCM I-2171, Bifidobacterium longum subsp. longum strain ATCC 15708, Bifidobacterium longum subsp. longum strain DSM 20097, Bifidobacterium longum subsp. longum strain NCIMB 8809, Bifidobacterium longum subsp. longum strain CNCM I-2618 (NCC 2705), Bifidobacterium longum subsp. longum strain CNCM I-2170, Bifidobacterium longum subsp. longum strain ATCC 15707 (T), or combinations thereof.

[0041] In some preferred embodiments, Bifidobacterium longum subsp. longum strain B. longum CNCM I-2618 (NCC 2705) is used.

[0042] In some embodiments, the fructooligosaccharide is selected from sucrose, lactulose, or combinations thereof. In some preferred embodiments, sucrose is used.

[0043] In some embodiments, the FOS has a DP in the range of 2 to 10, preferably 2 to 8. BRIEF DESCRIPTION OF THE DRAWINGS

[0044]

Figure 1

Figure 2

Figure 3

Figure 4

[0045] [MODE FOR CARRYING OUT THE INVENTION] Composition The composition of the present invention may be in the form of a food, a medical food, enteral nutrition, a nutritional composition, or a nutritional supplement. The term "nutritional supplement" refers to a product intended to supplement the normal diet of a subject.

[0046] In one embodiment, the food is selected from milk, yogurt, kefir, cheese, fermented milk, milk-based fermented products, rice-based products, milk-based powders, infant formulas, and pet foods.

[0047] The composition may be in the form of a medical food. As used herein, the term "medical food" refers to a food product specially formulated for the dietary management of a medical disease or condition. A medical food may be administered under medical supervision. A medical food may be for oral intake or enteral nutrition.

[0048] The composition may be in the form of enteral nutrition. The term "enteral nutrition" refers to products intended to introduce nutrition directly into the gastrointestinal tract of a subject through a feeding tube. Enteral nutrition may be administered, for example, through a feeding tube placed through the subject's nose (such as a nasogastric tube, a nasoduodenal tube, and a nasojejunal tube), or through a feeding tube placed directly into the subject's abdomen (such as a gastrostomy feeding tube, a gastrojejunostomy feeding tube, or a jejunostomy feeding tube).

[0049] The composition may be in the form of a pharmaceutical composition and may contain one or more suitable carriers, diluents, and / or additives acceptable as a medicine.

[0050] Examples of such suitable additives for the compositions described herein can be found in "Handbook of Pharmaceutical Excipients", 2nd Edition (1994), edited by A Wade and PJ Weller.

[0051] Carriers or diluents acceptable for therapeutic use are known in the pharmaceutical field and are described, for example, in "Remington’s Pharmaceutical Sciences", Mack Publishing Co. (A.R. Gennaro edit. 1985).

[0052] Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, etc. Examples of suitable diluents include ethanol, glycerol, and water.

[0053] In selecting a pharmaceutical carrier, additive, or diluent, it can be selected in relation to the intended route of administration and standard pharmaceutical operations. The pharmaceutical composition may contain, as a carrier, additive, or diluent, or in addition thereto, any suitable binder, lubricant, suspending agent, coating agent, and / or solubilizing agent.

[0054] Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, flowable lactose, β-lactose, etc., corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethyl cellulose, and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc.

[0055] The composition may also contain preservatives, stabilizers, dyes, and even flavoring agents. Examples of preservatives include sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents can also be used.

[0056] Nutritionally acceptable carriers, diluents, and additives include those suitable for human or animal ingestion as standards in the food industry. Typical nutritionally acceptable carriers, diluents, and additives are well known to those skilled in the art.

[0057] The composition can be in the form of tablets, coated tablets, medicinal candies, capsules, gel caps, powders, granules, solutions, emulsions, suspensions, coated particles, spray-dried particles, or pills.

[0058] In an alternative embodiment, the composition may be in the form of a topical composition such as a gel, cream, ointment, emulsion, suspension, or solution for topical administration.

[0059] It will be apparent to those skilled in the art that the ideal dosage will vary, for example, depending on the subject to be treated, their state of health, gender, age, or weight, and the route of administration. As a result, the dosage that is ideally used can vary, but can be readily determined by those skilled in the art.

[0060] However, generally, when the composition of the present invention contains 10 6 ~10 10 cfu, and / or 10 6 ~10 10 cells of Bifidobacterium longum subsp. longum per daily dosage, it is preferable. The composition of the present invention may also contain 10 6 ~10 11 cfu, and / or 10 6 ~10 11 cells of Bifidobacterium longum subsp. longum per 1 g of the dry weight of the composition.

[0061] Bifidobacterium longum Genes encoding serpins and their surroundings are highly conserved within Bifidobacterium longum subsp. longum. Bifidobacterium longum may be any Bifidobacterium longum subsp. longum strain. In some embodiments, the Bifidobacterium longum subsp. longum strain is Bifidobacterium longum subsp. longum strain CNCM I-2169, Bifidobacterium longum subsp. longum strain CNCM I-2171, Bifidobacterium longum subsp. longum strain ATCC BAA-999 (available as BB536 from Morinaga Milk Industry Co., Ltd.), Bifidobacterium longum subsp. longum strain ATCC15708, Bifidobacterium longum subsp. longum strain DSM 20097, Bifidobacterium longum subsp. longum strain NCIMB 8809, Bifidobacterium longum subsp. longum strain CNCM I-2618 (NCC 2705), Bifidobacterium longum subsp. longum strain CNCM I-2170, Bifidobacterium longum subsp. longum strain ATCC 15707 (T), Bifidobacterium longum subsp. longum strain CNCM I-103, Bifidobacterium longum subsp. longum strain CNCM I-2334, Bifidobacterium longum subsp. longum strain CNCM I-3864, Bifidobacterium longum subsp. longum strain CNCM I-3853, or a combination thereof. The strains are deposited with the depository institutions shown in the table below (Table 1) and are assigned the following deposit dates and accession numbers:

[0062]

Table 1

[0063] CNCM refers to the Collection nationale de cultures de micro-organismes (Pasteur Institute, 28, rue du Dr Roux, F-75724 Paris Cedex 15, France). ATCC refers to the American Type Culture Collection (10801 University Blvd., Manassas, Virginia 20110-2209, U.S.A.). DSM refers to the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures (Inhoffenstr.7B, D-38124 Braunschweig, Germany). NCIMB refers to NCIMB Ltd (Ferguson Building, Craibstone Estate, Buckburn, Aberdeen AB21 9YA, Scotland).

[0064] Strains 1, 2, 6, 7, 9, 11 to 13 were deposited by Nestec S.A. (avenue Nestle 55, 1800 Vevey, Switzerland). Subsequently, since Nestec S.A. was merged into Societe des Produits Nestle S.A., according to Article 2(ix) of the Budapest Treaty, Societe des Produits Nestle S.A. is the successor in title of Nestec S.A. All other strains are commercially available.

[0065] In some preferred embodiments, Bifidobacterium longum subsp. longum may be selected from Bifidobacterium longum subsp. longum strain CNCM I-2169, Bifidobacterium longum subsp. longum strain CNCM I-2171, Bifidobacterium longum subsp. longum strain ATCC 15708, Bifidobacterium longum subsp. longum strain DSM 20097, Bifidobacterium longum subsp. longum strain NCIMB 8809, Bifidobacterium longum subsp. longum strain CNCM I-2618 (NCC 2705), Bifidobacterium longum subsp. longum strain CNCM I-2170, Bifidobacterium longum subsp. longum strain ATCC 15707 (T), or combinations thereof.

[0066] In some preferred embodiments, Bifidobacterium longum subsp. longum strain B. longum CNCM I-2618 (NCC 2705) is used.

[0067] Fructose disaccharide and FOS The inventors have surprisingly found that fructose disaccharide and fructooligosaccharides (FOS) can increase the production of serpins in bacteria of the Bifidobacterium longum species, subspecies longum.

[0068] As used herein, the term "oligosaccharide" refers to a carbohydrate having a degree of polymerization (DP) in the range of 2 to 20 (including the values at both ends).

[0069] "Degree of polymerization" or "DP" refers to the total number of sugar units in a chain of an oligosaccharide or polysaccharide.

[0070] As used herein, the term "fructose disaccharide" refers to a disaccharide containing at least one fructose molecule.

[0071] Examples of suitable fructose disaccharides include sucrose (composed of one glucose molecule and one fructose molecule), lactulose (composed of one galactose and one fructose molecule), and turanose (composed of glucose and one fructose molecule).

[0072] As used herein, the term "fructooligosaccharide" (FOS) refers to indigestible oligosaccharides containing two or more fructose molecules. The fructooligosaccharides of the present invention have a DP of 2 to 200, preferably 2 to 100, such as 2 to 60, more preferably 2 to 20, or 2 to 10. In some preferred embodiments, the FOS is short-chain FOS and has a DP in the range of 2 to 10 (including both end values), preferably in the range of 2 to 8 (including both end values).

[0073] Based on the monomeric subunits, preferably at least 30%, preferably at least 60%, more preferably at least 90% of the sugar units of FOS are fructose units. In some preferred embodiments, the FOS comprises fructose monomeric subunits linked by β-1,2 bonds, optionally together with terminal glucose subunits. Preferably, it has a DP of 2 to 8 (including both end values).

[0074] Due to the structure of its glycosidic bonds, fructooligosaccharide (FOS) is mostly resistant to hydrolysis by salivary and intestinal digestive enzymes. FOS is classified as a prebiotic, an indigestible carbohydrate that confers beneficial effects on the host by stimulating the growth and / or activity of beneficial bacteria in the colon.

[0075] Bifidobacterium longum subsp. longum may be cultured in a medium containing raffinose or FOS, or a mixture thereof, for example, at a concentration of 0.02 to 5% by weight. For example, Bifidobacterium longum subsp. longum may be cultured in a medium containing raffinose or FOS, or a mixture thereof, at a concentration of 0.02 to 5% by weight, 0.05 to 2% by weight, 0.1 to 1.5% by weight, or about 1% by weight.

[0076] Raffinose or FOS, or a mixture thereof, may be added to a conventional culture medium containing up to 8% by weight, preferably up to 6% by weight, for example up to 4% by weight, of another sugar suitable for maintaining the growth of B. bacterium longum, such as, but not limited to, glucose. Raffinose can induce the production of serpins in Bifidobacterium longum subsp. longum even in the presence of glucose. Preferably, the culture medium at the end of fermentation contains less than 0.4% by weight of glucose, such as 0% to 0.3% by weight of glucose. Conventional culture media suitable for the growth of B. longum are well known to those skilled in the art.

[0077] In one embodiment, Bifidobacterium longum subsp. longum may be cultured in a medium containing raffinose or FOS at a concentration of 0.05 to 2% by weight, 0.1 to 1.5% by weight, or about 1% by weight, optionally in the presence of glucose. Preferably, the culture medium at the end of fermentation contains less than 0.4% by weight of glucose, such as 0% to 0.3% by weight of glucose.

[0078] In one embodiment, sucrose is used at the above concentrations.

[0079] In one embodiment, FOS is used at the above concentrations.

[0080] Method for producing a culture powder B. Grow the strains belonging to the genus Longum under anaerobic conditions. Fermentation methods under anaerobic conditions are well known. Those skilled in the art can identify suitable components of the fermentation medium and adjust the fermentation conditions based on their general knowledge according to the microorganism to be grown. The fermentation medium typically comprises a nitrogen source such as yeast extract, and a carbon source such as sugar, and various growth factors necessary for the microorganism (e.g., minerals, vitamins, etc.), and water.

[0081] A non-limiting example of a typical growth medium for B. Longum is MRS (De Man, Rogosa and Sharpe) medium (MRSc) supplemented with 0.05% cysteine.

[0082] The fermentation is preferably carried out in two steps, with a starter fermentation being carried out before the main fermentation step. The fermentation medium may be the same or different for the starter fermentation and the main fermentation.

[0083] The second step of this method is the concentration of the biomass. This can also be carried out using methods known to those skilled in the art, such as centrifugation or filtration. The total solids of the concentrated biomass preferably constitute 10 - 35% by weight, preferably 14 - 35% by weight, based on the total dry weight of the biomass (i.e., the total amount of the fermentation medium and the prepared microorganism).

[0084] Optionally, the concentration may precede or be combined with a washing step to remove residues of the fermentation medium and / or compounds generated during fermentation. For example, the washing can be carried out by concentrating the biomass, resuspending the concentrated biomass in a buffer such as a phosphate buffer or a similar composition, and re-concentrating the biomass.

[0085] For example, the method described in International Publication No. WO 2017 / 001590, which is incorporated herein by reference in its entirety, can be applied.

[0086] Combination According to one aspect of the present invention, a combination of (i) Bifidobacterium longum subsp. longum and (ii) fructooligosaccharide or FOS, or a combination thereof, is provided.

[0087] As used herein, the term "combination" refers to the combined administration of Bifidobacterium longum subsp. longum with fructooligosaccharide or FOS, or a combination thereof, and Bifidobacterium longum subsp. longum and fructooligosaccharide and / or FOS may be administered simultaneously or sequentially.

[0088] As used herein, the terms "simultaneous" or "simultaneously" are used to mean that two agents are administered at the same time, i.e., at the same moment.

[0089] The terms "sequential" or "sequentially" are used to mean that two agents are administered one after the other, where Bifidobacterium longum subsp. longum and fructooligosaccharide or FOS, or a combination thereof, may be administered first.

[0090] The agents may be administered either as separate formulations or as a single combined formulation.

[0091] When the compounds are co-formulated, i.e., formulated in the same composition or formulation, they can only be administered simultaneously. When the compounds are formulated in separate compositions or formulations, they can be administered simultaneously or sequentially. The simultaneous administration of the agents in the same formulation or separate formulations can also be described as the co-administration or joint-administration of the two compounds.

[0092] In one embodiment, Bifidobacterium longum subsp. longum and fructooligosaccharide or FOS, or a combination thereof, are a mixture. In another embodiment, Bifidobacterium longum subsp. longum and fructooligosaccharide or FOS, or a combination thereof, are present in the form of a kit that includes a preparation of these two agents and, optionally, instructions for the simultaneous or sequential administration of the preparation to a subject in need thereof.

[0093] Treatment The Bifidobacterium longum subsp. longum strain prepared according to the present invention, or a composition containing the same, can be used for the treatment or prevention of gluten-related disorders or conditions accompanied by a decrease in the activity of serine protease inhibitors.

[0094] For example, the Bifidobacterium longum subsp. longum prepared according to the present invention, or a composition containing the same, can be used for the treatment or prevention of inflammatory bowel disease, celiac disease, non-celiac gluten sensitivity, gluten ataxia, dermatitis herpetiformis, and wheat allergy.

[0095] Preferably, the disease is a gluten-related disorder. Gluten-related disorders include diseases induced by gluten. The terms "conditions related to gluten sensitivity" and "gluten-related disorders" are used interchangeably herein. Examples of gluten-related disorders include celiac disease, non-celiac gluten sensitivity, gluten ataxia, dermatitis herpetiformis, and wheat allergy.

[0096] Celiac disease Celiac disease is one of the most common immune-mediated disorders. This disorder is seen worldwide, particularly prominent in the United States and Europe, with approximately 1% of the tested population being positive in antibody tests. Celiac disease is a complex disorder resulting from the complex interactions among various immunological, genetic, and environmental factors. This disease is induced by the digestion of wheat gluten and other related cereal proteins such as rye protein and barley protein. Symptoms associated with celiac disease include growth retardation, irritability, and delayed puberty in children, as well as many gastrointestinal symptoms such as discomfort, diarrhea, latent stools, steatorrhea, and flatulence.

[0097] Clinical evidence has shown that class II human leukocyte antigen (HLA-DQII), which is strongly associated with the pathological conditions of celiac disease, is expressed in approximately 95% of celiac disease patients. In the intestinal lumen, gluten proteins are partially digested to form 33-mer gluten peptides that are resistant to proteolysis. After passing through the small intestinal barrier, these peptides are deamidated by negatively charged transglutaminase 2 (TG2) (Sollid, 2000, Annual review of immunology, 18(1), 53-81), and then bind to the positively charged binding sites of HLA-DQ2.5 / 8 (Dieterich et al., 1997, Nature medicine, 3(7), 797-801). HLA-DQ2.5 / 8 presents these specific gluten peptide signals to helper T cells, and other immune cells cause further damage in the small intestine. Antibodies against gluten proteins and autoantibodies against connective tissue components (TG2) are also associated with the progression of celiac disease (Alaedini & Green, 2005, Annals of internal medicine, 142(4), 289-298).

[0098] Non-celiac gluten sensitivity Non-celiac gluten sensitivity (also called non-celiac wheat sensitivity) is a newly emerging condition. This condition is defined as a clinical entity that causes intestinal and / or extra-intestinal symptoms induced by gluten intake and may be improved by removing gluten-containing foods from the diet (Lundin & Alaedini, 2012). The etiology of non-celiac gluten sensitivity has not yet been fully elucidated. It has been shown that other proteins / peptides in gluten and gluten-containing grains (wheat, rye, barley, and their derivatives) other than gliadin (the major cytotoxic antigen of gluten) may be involved in the appearance of symptoms. Non-celiac gluten sensitivity is the most common syndrome among gluten-related disorders, showing a prevalence of 0.5 - 13% in the general population (Catassi et al., 2013, Nutrients, 5(10), 3839 - 385). The diagnosis of non-celiac gluten sensitivity is made by excluding other gluten-related disorders.

[0099] Dermatitis herpetiformis Dermatitis herpetiformis is a chronic autoimmune blistering skin disease characterized by the presence of skin lesions that have a widespread and symmetrical distribution, are predominant in areas of high friction, mainly affect both elbows, knees, buttocks, and ankles, and can also affect the scalp and other parts of the body. The lesions are covered with vesicular crusts, and when they peel off, they develop into areas of pigmentation, or chronic intense burning, itching, and vesicular eruptions.

[0100] The age of onset varies. It can start in childhood and adolescence, but can affect any age and either sex throughout a person's life.

[0101] People with dermatitis herpetiformis have various degrees of intestinal lesions ranging from moderate mucosal lesions to the presence of villous atrophy.

[0102] Wheat allergy The gastrointestinal symptoms of wheat allergy are similar to those of celiac disease and non-celiac gluten sensitivity, but the interval from exposure to wheat to the onset of symptoms is different. Wheat allergy has an early onset (from a few minutes to a few hours) after ingestion of wheat-containing foods and can lead to anaphylaxis.

[0103] Gluten ataxia Gluten ataxia is a gluten-related disorder. In gluten ataxia, damage occurs in the cerebellum, i.e., the balance center of the brain that controls coordinated movements and complex movements such as walking, speech, and swallowing. Gluten ataxia is one of the most common causes among sporadic idiopathic ataxias. This ataxia accounts for 40% of ataxias of unknown cause and 15% of all ataxias.

[0104] Gluten ataxia is an immune-mediated disease induced by gluten ingestion in genetically hypersensitive individuals. It is a disease that should be considered in the differential diagnosis of all patients with idiopathic sporadic ataxia. The effectiveness of treatment depends on the elapsed time from the onset of ataxia to diagnosis. The neuron death in the cerebellum resulting from the exposure of the subject to gluten is irreversible.

[0105] Early diagnosis and treatment with a gluten-free diet can improve ataxia and prevent its progression. Less than 10% of people with gluten ataxia present with any gastrointestinal symptoms, but about 40% have intestinal damage. Anti-gliadin antibodies are mentioned as susceptibility markers for gluten ataxia. Immunoglobulin A (IgA) deposited against transglutaminase 2 (TG2) in the small intestine and extraintestinal sites has been proven to be even more reliable.

[0106] Administration Bifidobacterium longum subsp. longum or the composition described herein is preferably administered enterally.

[0107] Enteral administration may be oral, intragastric, and / or rectal administration.

[0108] Generally speaking, the administration of the combinations or compositions described herein may be, for example, administration to the gastrointestinal tract by the oral route or another route, and for example, the administration may be by enteral nutrition.

[0109] In an alternative embodiment, the administration of the combinations or compositions described herein may be topical administration.

[0110] The subject may be a mammal such as a human, dog, cat, horse, goat, cow, sheep, pig, deer, and primate. Preferably, the subject is a human.

[0111] The preferred features and embodiments of the present invention will now be described by way of non-limiting examples.

[0112] In the practice of the present invention, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology, and immunology, which are within the capabilities of those skilled in the art, are used. Such techniques are described in the literature. For example, see Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, J.M. and McGee, J.O’D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M.J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press, and Lilley, D.M. and Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is incorporated herein by reference.

[0113] [Examples] Example 1 - Serpin induction of B. longum CNCM I-2618 (NCC 2705) by sucrose B. longum strain CNCM I-2618 (NCC 2705) was grown in a Biolector (growth conditions - anaerobic, 37 °C) on a sugar-free MRS + 5 mM L-cysteine (MRSc) base supplemented with various carbohydrates.

[0114] The strain was cultured in a Biolector (m2p-labs Aachen, Germany) using a 48-well microtiter plate in combination with a pH sensor and a dissolved oxygen (DO) sensor. To prevent bacterial aggregation, the plate was shaken continuously for 24 and 48 h. The cultures were harvested by centrifugation and the supernatant was removed. The pellet was resuspended in PBS supplemented with Halt protease inhibitor (Sigma) and lysed using glass beads. A lysate containing both soluble and insoluble material was then recovered. The total protein content was measured using a Pierce BCA kit (Thermofisher) and the serpin protein concentration was determined using ELISA.

[0115] As shown in Figure 1, it was revealed that the monosaccharide fructose increased the B. longum NCC 2705 serpin protein level compared to all other sugars tested.

[0116] As shown in Figure 2, fructose was able to induce serpin in B. longum NCC2705 to a higher level than galactose. However, this strain requires more time to switch to a fructose-based regime, resulting in a growth delay (indicated by the triangles representing the culture density measured by the optical density at 600 nm), and thus requires more time to reach the same level of serpin protein compared to when grown on galactose.

[0117] Example 2 - Serpin induction of B. longum CNCM I-2618 (NCC 2705) by fructose disaccharide in the presence of glucose B. longum NCC 2705 was cultured in a sugar-free MRSc base (as described in Example 1) in a Biolector with various disaccharides and trisaccharides added at concentrations of 0.5 wt% and 1 wt%. After 48 hours of growth, the cultures were harvested and the total protein level and the serpin protein level were analyzed (as described in Example 1).

[0118] Figure 3 shows that fructose disaccharides and trisaccharides can induce serpin production to higher levels than the other disaccharides tested. In the case of raffinose (a trisaccharide), a significant increase in serpin levels was obtained only at a concentration of 0.5 wt% compared to 1 wt%. Without being bound by any theory, this is thought to be because when the trisaccharide is used at 1 wt%, a high level of residual glucose is present at the end of fermentation. Surprisingly, it was found that sucrose can strongly induce serpin production even at a concentration of 1 wt%.

[0119] Example 3 - Serpin induction of B. longum CNCM I-2618 (NCC 2705) by short-chain fructooligosaccharides (FOS) B. longum NCC 2705 was cultured in a sugar-free MRSc base (as described in Example 1) in a Biolector with various short-chain fructooligosaccharides (FOS) added at a concentration of 1 wt%. The cultures were harvested after 16 hours of growth and the total protein level and the serpin protein level were analyzed (as described in Example 1).

[0120] The FOS used were: (i) NutraFlora P95 FO from Ingredion Inc, USA (a short-chain FOS having glucose-fructose (GF) chains with a DP of 3 - 5 and containing GF2, GF3, and GF4 molecules); (ii) Orafti P95 from Beneo (a short-chain FOS with a DP of 2 - 8).

[0121] Figure 4 shows that FOS can induce serpin production to higher levels than the growth control with glucose.

Claims

1. Use of fructooligosaccharide, fructooligosaccharide (FOS), or a combination thereof to increase the production of serpintin protein in Bifidobacterium longum subsp. longum.

2. The use according to claim 1, wherein the Bifidobacterium longum subsp. longum is cultured in a medium containing the fructooligosaccharide, fructooligosaccharide (FOS), or a combination thereof at a concentration of 0.02 to 5% by weight.

3. A method for increasing the level of serpintin protein in Bifidobacterium longum subsp. longum, the method comprising the step of growing Bifidobacterium longum subsp. longum in a culture medium, wherein the culture medium contains fructooligosaccharide, FOS, or a combination thereof.

4. The method according to claim 3, wherein the culture medium contains the fructooligosaccharide, FOS, or a combination thereof at a concentration of 0.02 to 5% by weight.

5. The Bifidobacterium longum subsp. longum is selected from the group consisting of Bifidobacterium longum subsp. longum strain CNCM I-2169, Bifidobacterium longum subsp. longum strain CNCM I-2171, Bifidobacterium longum subsp. longum strain ATCC BAA-999, Bifidobacterium longum subsp. longum strain ATCC 15708, Bifidobacterium longum subsp. longum strain DSM 20097, Bifidobacterium longum subsp. longum strain NCIMB 8809, Bifidobacterium longum subsp. longum strain CNCM I-2618 (NCC 2705), Bifidobacterium longum subsp. longum strain CNCM I-2170, Bifidobacterium longum subsp. longum strain ATCC 15707 (T), or a combination thereof, the use according to claim 1 or 2, or the method according to claim 3 or 4.

6. The Bifidobacterium longum subsp. longum is Bifidobacterium longum strain CNCM I-2618 (NCC 2705), the use according to claim 1, 2 or 5, or the method according to claim 3, 4 or 5.

7. Use according to any one of claims 1, 2, 5 or 6, or a method according to any one of claims 3 to 6, wherein the fructooligosaccharide or FOS is selected from sucrose, lactulose, and combinations thereof.

8. Use according to any one of claims 1, 2, 5 or 6, or a method according to any one of claims 3 to 6, wherein the fructooligosaccharide or FOS is FOS having a DP in the range of 2 to 10.

9. Bifidobacterium longum subsp. longum prepared by a method of growing Bifidobacterium longum subsp. longum in a culture medium, wherein the culture medium contains fructooligosaccharide, FOS, or a combination thereof.

10. Bifidobacterium longum subsp. longum prepared by the method according to claim 9, wherein the culture medium contains the fructooligosaccharide, FOS, or a combination thereof at a concentration of 0.02 to 5% by weight.

11. The Bifidobacterium longum subsp. longum is selected from Bifidobacterium longum subsp. longum strain CNCM I-2169, Bifidobacterium longum subsp. longum strain CNCM I-2171, Bifidobacterium longum subsp. longum strain ATCC BAA-999 (available as BB536 from Morinaga Milk Industry Co., Ltd.), Bifidobacterium longum subsp. longum strain ATCC 15708, Bifidobacterium longum subsp. longum strain DSM 20097, Bifidobacterium longum subsp. longum strain NCIMB 8809, Bifidobacterium longum subsp. longum strain CNCM I-2618 (NCC 2705), Bifidobacterium longum subsp. longum strain CNCM I-2170, Bifidobacterium longum subsp. longum strain ATCC 15707 (T), Bifidobacterium longum subsp. longum strain CNCM I-103, Bifidobacterium longum subsp. longum strain CNCM I-2334, Bifidobacterium longum subsp. longum strain CNCM I-3864, Bifidobacterium longum subsp. longum strain CNCM I-3853, or a combination thereof, and is prepared by the method according to claim 9 or 10.

12. A composition comprising Bifidobacterium longum subsp. longum prepared by the method according to any one of claims 9 to 11.

13. For use in the treatment or prevention of inflammatory bowel disease, celiac disease, non-celiac gluten sensitivity, gluten ataxia, dermatitis herpetiformis, or wheat allergy, Bifidobacterium longum subsp. longum prepared by the method according to any one of claims 9 to 11, or the composition according to claim 12.

14. For use in the treatment or prevention of inflammatory bowel disease, celiac disease, non-celiac gluten sensitivity, gluten ataxia, dermatitis herpetiformis, or wheat allergy, a combination of (i) Bifidobacterium longum subsp. longum and (ii) fructooligosaccharide, FOS, or a combination thereof.

15. The combination for use according to claim 14, wherein the Bifidobacterium longum subsp. longum is selected from Bifidobacterium longum subsp. longum strain CNCM I-2169, Bifidobacterium longum subsp. longum strain CNCM I-2171, Bifidobacterium longum subsp. longum strain ATCC BAA-999 (available as BB536 from Morinaga Milk Industry Co., Ltd.), Bifidobacterium longum subsp. longum strain ATCC 15708, Bifidobacterium longum subsp. longum strain DSM 20097, Bifidobacterium longum subsp. longum strain NCIMB 8809, Bifidobacterium longum subsp. longum strain CNCM I-2618 (NCC 2705), Bifidobacterium longum subsp. longum strain CNCM I-2170, Bifidobacterium longum subsp. longum strain ATCC 15707 (T), Bifidobacterium longum subsp. longum strain CNCM I-103, Bifidobacterium longum subsp. longum strain CNCM I-2334, Bifidobacterium longum subsp. longum strain CNCM I-3864, Bifidobacterium longum subsp. longum strain CNCM I-3853, or a combination thereof.

16. The combination according to claim 14 or 15, wherein the fructooligosaccharide or FOS is selected from sucrose, lactulose, or a combination thereof.

17. The combination according to claim 14 or 15, wherein the fructooligosaccharide or FOS is an FOS having a DP in the range of 2 to 10.

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