Serpin production

Abstract

Use of galactose or galactooligosaccharides to increase serpin protein production in Bifidobacterium longum subsp. longum.

Description

[Technical Field]

[0001] The present invention relates to bacteria that express serpins, methods for increasing serpin production in bacteria, and uses thereof.

[0002] [Background technology] Gluten-related disorders include all diseases induced by gluten. These include, among other pathophysiologies, celiac disease and non-celiac gluten sensitivity. Currently, the incidence of various gluten-related disorders is increasing worldwide, especially for celiac disease and non-celiac gluten sensitivity. Both diseases are induced by the ingestion of gluten. Both innate 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 the gold standard for treating celiac disease and non-celiac gluten sensitivity, but it may have limitations in addressing extraintestinal manifestations of the disease (Sedghizadeh et al., 2002, Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology, 94(4), 474-478). Following a strict gluten-free diet has proven challenging because low-level cross-contamination is difficult to avoid and can occur throughout the food production chain, from grain growing to processing (Mitchison et al., 1991, Gut, 32(3), 260-265). Furthermore, it has been reported that even those on a strict gluten-free diet may unintentionally ingest up to 3 g of gluten daily (Aziz et al., 2014, The American Journal of Gastroenterology, 109(9), 1498).

[0004] Celiac disease is widespread, particularly in the United States and Europe, where approximately 1% of individuals tested positive for antibodies (Dube et al., 2005, Gastroenterology, 128(4), S57-S67). Celiac disease is a complex disorder resulting from a complex interaction between various immunological, genetic, and environmental factors (Alaedini & Green, 2005). The 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 irritability, diarrhea, occult stools, 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 extraintestinal symptoms induced by gluten ingestion and may improve with the elimination of 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 grains (wheat, rye, barley, and their derivatives) may be involved in the manifestation of symptoms. Non-celiac gluten sensitivity is the most common syndrome among gluten-related disorders, with a prevalence of 0.5–13% (average 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 strongly implicated in gluten-related disorders. Elafin is a human serine protease inhibitor that exhibits potent inhibitory activity against various forms of elastase and proteinases (Ying & Simon, 1993, Biochemistry, 32(7), 1866-1874). Elafin is expressed throughout the gastrointestinal epithelium, and its expression and induction are reduced in patients with inflammatory bowel disease and celiac disease (Baranger, Zani, Labas, Dallet-Choisy, & Moreau, 2011; Motta et al., 2012). Recently, elafin has been identified as a substrate for the cross-linking activity of transglutaminase 2 (TG2) (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 therefore prevents the deamidation of the digestion-resistant 33-mer gliadin peptide, 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 pathology and normalize intestinal inflammation in a mouse model of gluten sensitivity (Galipeau et al., 2014, The American journal of gastroenterology, 109(5), 748-756). However, this proposed therapy is based on genetically modified organisms (GMOs), which makes it unsuitable for food applications due to the very low consumer acceptance of GMOs.

[0009] More recently, prokaryotic serpins have been reported. In silico analysis revealed the presence of genes encoding serpin-like proteins in various Bifidobacterium species, particularly in the bacterium Bifidobacterium longum subsp. longum. The protein encoded by B. longum subsp. longum (named B. longum) NCC 2705 exhibited antiprotease 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 is assigned the accession number CNCM I-2618.

[0011] Recently, it has been shown that B. longum NCC 2705 (CNCM I-2618) can ameliorate gluten-induced pathophysiology in a mouse model of gluten hypersensitivity through serpin production, 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 present inventors have surprisingly found that the addition of galactose and galactooligosaccharides (GOS) to the growth medium of Bifidobacterium longum subsp. longum bacteria can increase the production of serpins.

[0013] Thus, in a first aspect of the present invention there is provided the use of galactose or galactooligosaccharides (GOS), or a combination thereof, to increase 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 species Bifidobacterium longum subsp. longum, the method comprising growing Bifidobacterium longum subsp. longum in a culture medium, the culture medium comprising galactose or galactooligosaccharides (GOS), or a combination thereof.

[0015] According to another aspect of the present invention, there is provided a bacterium of the species Bifidobacterium longum subsp. longum produced by a method of growing Bifidobacterium longum subsp. longum in a culture medium, wherein the culture medium comprises galactose or GOS, or a combination thereof.

[0016] Bifidobacterium longum subsp. longum prepared according to the present invention is associated with increased serpin protein levels compared to the same Bifidobacterium longum subsp. longum strain grown in the absence of galactose or GOS, or a combination thereof.

[0017] According to the present invention, the Bifidobacterium longum subsp. longum may be cultured in a medium containing galactose or GOS, or a combination thereof, at a concentration of, for example, 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 galactose or GOS, or a combination thereof, at a concentration of 0.02 to 5 wt %, 0.05 to 2 wt %, 0.1 to 1.5 wt %, or about 1 wt %.

[0019] According to another aspect of the present invention, there is provided a composition comprising Bifidobacterium longum subsp. longum prepared by the methods described herein.

[0020] In one embodiment, the composition is a food, a medical food, a tube feeding, or a nutritional supplement.

[0021] In one embodiment, the food product is selected from milk, yogurt, curd, cheese, fermented milk, fermented milk-based products, rice-based products, milk-based powders, infant formula, and pet food.

[0022] In one embodiment, the composition is a pharmaceutical composition, which comprises one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0023] According to another aspect of the present invention there is provided Bifidobacterium longum subsp. longum prepared by the methods described herein, or a composition comprising said Bifidobacterium longum subsp. longum, for use in the treatment or prevention of a condition associated with gluten sensitivity or a condition associated with decreased activity of a serine protease inhibitor.

[0024] According to another aspect of the present invention there is provided Bifidobacterium longum subsp. longum prepared by a method described herein, or a composition comprising said Bifidobacterium longum subsp. longum, for use in the treatment or prevention of a gluten-related disorder.

[0025] According to one aspect of the present invention there is provided Bifidobacterium longum subsp. longum prepared by a method described herein, or a composition comprising said Bifidobacterium longum subsp. longum, for use in the treatment or prevention of celiac disease, non-celiac gluten sensitivity, gluten ataxia, dermatitis herpetiformis, or wheat allergy.

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

[0027] Bifidobacterium longum subsp. longum may be any Bifidobacterium longum subsp. longum strain. 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, in particular B. longum CNCM I-2618 (NCC 2705).

[0028] It will also be appreciated that when galactose or GOS, or a combination thereof, is administered in combination with Bifidobacterium longum subsp. longum, galactose and / or GOS may also increase serpin production in Bifidobacterium longum subsp. longum in vivo.

[0029] Thus, according to another aspect of the present invention, there is also provided a combination of (i) Bifidobacterium longum subsp. longum and (ii) galactose or GOS, or a combination thereof.

[0030] According to another aspect of the present invention, there is also provided a combination of (i) Bifidobacterium longum subsp. longum and (ii) galactose or GOS, or a combination thereof, for use in the treatment or prevention of a condition associated with gluten sensitivity or a condition associated with reduced levels of a serine protease inhibitor.

[0031] In one embodiment, the combination is B. longum strain CNCM I-2618 in combination with galactose.

[0032] According to another aspect of the present invention there is also provided Bifidobacterium longum subsp. longum for use in the treatment or prevention of a condition associated with gluten sensitivity or a condition associated with reduced levels of a serine protease inhibitor, wherein the Bifidobacterium longum subsp. longum is administered in combination with galactose or GOS, or a combination thereof.

[0033] According to another aspect of the present invention there is provided galactose or GOS, or a combination thereof, for use in the treatment or prevention of a condition associated with gluten sensitivity or a condition associated with reduced levels of a serine protease inhibitor, wherein the galactose or GOS, or a combination thereof, is administered in combination with Bifidobacterium longum subsp. longum.

[0034] 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.

[0035] 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 a combination thereof.

[0036] In some preferred embodiments, the Bifidobacterium longum subsp. longum strain B. longum CNCM I-2618 (NCC 2705) is used. [Brief explanation of the drawings]

[0037] [Figure 1] Serpin protein levels measured in B. longum NCC 2705 grown for 8 hours on various carbohydrates are shown. [Figure 2] Serpin protein levels measured in B. longum NCC 2705 grown on different ratios of glucose and galactose are shown. [Figure 3] Serpin protein levels measured in B. longum NCC 2705 grown in GOS for 8 hours are shown. [Figure 4] 1 shows the effect of (partially hydrolyzed guar gum (PHGG)) on serpin levels in B. longum NCC 2705. [Figure 5] Figure 1 shows the effect of galactose on serpin levels in B. longum subsp. longum strains capable of growth on galactose. Values represent protein levels normalized by the total amount of protein in each sample. [Figure 6]Figure 1 shows the effect of galactose on serpin levels in a B. longum subsp. longum strain that cannot grow on galactose alone. Values represent protein levels normalized by the total amount of protein in each sample. [Figure 7] Figure 1 shows the effects of galactose, GOS, and papain on various bifidobacterial strains harboring serpin-encoding genes. Values represent protein levels normalized by the total amount of protein in each sample.

[0038] [Mode for Carrying Out the Invention] composition The compositions of the present invention may be in the form of a food, a medical food, a tube feeding, a nutritional composition, or a nutritional supplement. The term "nutritional supplement" refers to a product intended to supplement a subject's normal diet.

[0039] In one embodiment, the food product is selected from milk, yogurt, curd, cheese, fermented milk, fermented milk-based products, rice-based products, milk-based powders, infant formula, and pet food.

[0040] 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 tube feeding.

[0041] The composition may be in the form of tube feeding. The term "tube feeding" refers to a product intended to introduce nutrition directly into the gastrointestinal tract of a subject through a feeding tube. Tube feeding 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).

[0042] The composition may be in the form of a pharmaceutical composition and may comprise one or more suitable pharmaceutically acceptable carriers, diluents and / or excipients.

[0043] Examples of such suitable excipients for the compositions described herein can be found in "Handbook of Pharmaceutical Excipients", 2nd Edition (1994), edited by A Wade and PJ Weller.

[0044] Acceptable carriers or diluents for therapeutic use are known in the pharmaceutical art and are described, for example, in "Remington's Pharmaceutical Sciences", Mack Publishing Co. (AR Gennaro edit. 1985).

[0045] Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol, etc. Examples of suitable diluents include ethanol, glycerol, and water.

[0046] The choice of pharmaceutical carrier, excipient, or diluent may be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient, or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), and / or solubilizing agent(s).

[0047] Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, fluid lactose, β-lactose, and the like, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like.

[0048] Preservatives, stabilizers, dyes, and even flavoring agents may be included in the composition. Examples of preservatives include sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents can also be used.

[0049] Nutritionally acceptable carriers, diluents, and additives include those suitable for human or animal consumption as specified in the food industry. Typical nutritionally acceptable carriers, diluents, and additives are well known to those skilled in the art.

[0050] The composition can be in the form of tablets, dragees, lozenges, capsules, gelcaps, powders, granules, solutions, emulsions, suspensions, coated particles, spray-dried particles, or pills.

[0051] In alternative embodiments, the composition may be in the form of a topical composition such as a gel, cream, ointment, emulsion, suspension, or solution for topical administration.

[0052] It will be apparent to one skilled in the art that the ideal dosage will vary depending on, for example, the subject being treated, its health condition, sex, age, or weight, and the route of administration. Consequently, the dosage ideally used may vary but can be readily determined by one skilled in the art.

[0053] However, in general, the compositions of the present invention are suitable for use in a daily dose of 10 6 ~10 10cfu, and / or cells 10 6 ~10 10 Preferably, the composition of the present invention contains 10 Bifidobacterium longum subsp. longum per gram of dry weight of the composition. 6 ~10 11 cfu, and / or cells 10 6 ~10 11 It may contain Bifidobacterium longum subsp. longum.

[0054] Bifidobacterium longum Bifidobacterium longum may be any Bifidobacterium longum subsp. longum strain. longum strain CNCM I-2169, Bifidobacterium longum subsp. longum strain CNCM I-2171, Bifidobacterium longum subsp. longum strain ATCC BAA-999 (available from Morinaga Milk Industry Co., Ltd. as BB536), 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 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.

[0055] The strains have been deposited at the depository institutions shown in the table below (Table 1) and have been assigned the following deposit dates and accession numbers:

[0056] [Table 1]

[0057] CNCM stands for Collection nationale de cultures de micro-organismes (Institut Pasteur, 28, rue du Dr Roux, F-75724 Paris Cedex 15, France). ATCC stands for American Type Culture Collection. (10801 University Blvd., Manassas, Virginia 20110-2209, USA). 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).

[0058] Strains 1, 2, 6, 7, 9, 11-13 have been deposited by Nestec SA (Avenue Nestle 55, 1800 Vevey, Switzerland). Nestec SA has since merged with Société des Produits Nestle SA, which is Nestec SA's successor in title pursuant to Article 2(ix) of the Budapest Treaty. All other strains are commercially available.

[0059] 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 a combination thereof.

[0060] In some preferred embodiments, the Bifidobacterium longum subsp. longum strain B. longum CNCM I-2618 (NCC 2705) is used.

[0061] GOS The present inventors have surprisingly found that galactose and galactooligosaccharides (GOS) are able to increase the production of serpins in bacteria of the species Bifidobacterium longum subsp. longum.

[0062] As used herein, the term "oligosaccharide" refers to carbohydrates having a degree of polymerization (DP) ranging from 2 to 20, inclusive.

[0063] "Degree of polymerization" or "DP" refers to the total number of sugar units in an oligosaccharide or polysaccharide chain.

[0064] The term "galactooligosaccharide" as used herein refers to an indigestible oligosaccharide containing two or more galactose molecules. The galactooligosaccharide of the present invention has a DP of 2 to 20, preferably 2 to 10. For example, based on the monomer subunit, preferably at least 30%, preferably at least 50%, more preferably at least 60% of the sugar units are galactose units.

[0065] Suitable galactooligosaccharides are commercially available, for example, Purimune GOS (from Corn Products International), King GOS (from King Prebiotics), Vivinal GOS (from Friesland Campina), and PHGG (from Taiyo). Other sources of oligosaccharides include Clasado, Ingredion, Leprino, Yakult, Dextra Laboratories, Sigma-Aldrich Chemie GmbH, and Kyowa Hakko Kogyo Co., Ltd. Alternatively, neutral oligosaccharides may be produced using specific glycosyltransferases, such as galactosyltransferases.

[0066] Galactooligosaccharides (GOS) are largely resistant to hydrolysis by saliva and intestinal digestive enzymes due to their glycosidic bond structure. GOS are classified as prebiotics, which are indigestible carbohydrates that confer beneficial effects on the host by stimulating the growth and / or activity of beneficial bacteria in the colon.

[0067] Bifidobacterium longum subsp. longum may be cultured in a medium containing galactose or GOS, or a mixture thereof, at a concentration of, for example, 0.02 to 5% by weight, such as 0.02 to 5%, 0.05 to 2%, 0.1 to 1.5%, or about 1% by weight.

[0068] Galactose or GOS, or a mixture thereof, may be added to a conventional culture medium containing up to 8% by weight, preferably up to 6% by weight, e.g., up to 4% by weight, of another sugar suitable for supporting the growth of B. longum, such as, but not limited to, glucose. The inventors surprisingly found that galactose can induce serpin production in Bifidobacterium longum subsp. longum, even in the presence of glucose, but only if the glucose is present at a level that can be depleted during fermentation. 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, e.g., 0.02% to 0.4% by weight, or about 0.05% to about 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.

[0069] In one embodiment, Bifidobacterium longum subsp. longum may be cultured in a medium containing galactose at a concentration of 0.05-2 wt%, 0.1-1.5 wt%, or about 1 wt%, optionally in the presence of glucose at a concentration that allows the galactose to be depleted by the end of fermentation. Preferably, the culture medium at the end of fermentation contains less than 0.4 wt%, such as 0-0.3 wt% glucose. If glucose is present, the culture medium may contain, for example, 0.02-0.4 wt%, or about 0.05-0.3 wt% glucose at the end of fermentation.

[0070] In one embodiment, Bifidobacterium longum subsp. longum may be cultured in a medium containing GOS at a concentration of 0.05-2%, 0.1-1.5%, or about 1% by weight, optionally in the presence of 0.02%-0.4%, or about 0.05%-0.3% residual glucose by weight.

[0071] In one embodiment, galactose is used at the concentrations described above.

[0072] In one embodiment, the GOS is used at the concentrations described above.

[0073] Culture powder manufacturing method Strains belonging to the species B. longum are grown 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 depending on the microorganism to be grown. The fermentation medium typically contains: a nitrogen source such as yeast extract; A carbon source such as sugar, Various growth factors necessary for microorganisms (e.g., minerals, vitamins, etc.) and water.

[0074] 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.

[0075] The fermentation is preferably carried out in two steps, with a starter fermentation carried out before the main fermentation step. The fermentation medium may be different or the same for the starter and main fermentations.

[0076] The second step of this method is the concentration of the biomass, which can also be carried out using methods known to those skilled in the art, such as centrifugation or filtration. The total solids content of the biomass after concentration preferably constitutes 10 to 35% by weight, preferably 14 to 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).

[0077] Optionally, concentration may be preceded or combined with a washing step to remove residues of the fermentation medium and / or compounds produced during fermentation. For example, washing may be performed by concentrating the biomass, resuspending the concentrated biomass in a buffer, such as a phosphate buffer or similar composition, and reconcentrating the biomass.

[0078] For example, the methods described in WO 2017 / 001590, which is incorporated by reference in its entirety, can be applied. combination According to one aspect of the present invention, there is provided a combination of (i) Bifidobacterium longum subsp. longum and (ii) galactose or GOS, or a combination thereof.

[0079] As used herein, the term "combination" refers to the combined administration of Bifidobacterium longum subsp. longum and galactose or GOS, or a combination thereof, wherein Bifidobacterium longum subsp. longum and galactose and / or GOS may be administered simultaneously or sequentially.

[0080] As used herein, the terms "concurrent" or "simultaneously" are used to mean that two agents are administered at the same time, i.e., at the same time.

[0081] The terms "sequential" or "sequentially" are used to mean that the two agents are administered one after the other, where either Bifidobacterium longum subsp. longum and galactose or GOS, or a combination thereof, can be administered first.

[0082] The agents may be administered either as separate formulations or as a single combined formulation.

[0083] When compounds are co-formulated, i.e., formulated in the same composition or formulation, they can only be administered simultaneously. When compounds are formulated in separate compositions or formulations, they can be administered simultaneously or sequentially. Simultaneous administration of agents in the same formulation or separate formulations can also be described as co-administration or joint-administration of two compounds.

[0084] In one embodiment, Bifidobacterium longum subsp. longum and galactose or GOS, or a combination thereof, are in a mixture. In another embodiment, Bifidobacterium longum subsp. longum and galactose or GOS, or a combination thereof, are present in the form of a kit comprising preparations of the two agents and, optionally, instructions for the simultaneous or sequential administration of the preparations to a subject in need thereof.

[0085] treatment The Bifidobacterium longum subsp. longum strain prepared according to the present invention, or a composition containing the same, can be used to treat or prevent gluten-related disorders or conditions associated with decreased activity of serine protease inhibitors.

[0086] For example, Bifidobacterium longum subsp. longum prepared according to the present invention, or a composition containing same, can be used to treat or prevent inflammatory bowel disease, celiac disease, non-celiac gluten sensitivity, gluten intoxication, dermatitis herpetiformis, and wheat allergy.

[0087] Preferably, the disease is a gluten-related disorder. Gluten-related disorders include diseases induced by gluten. The terms "conditions related to gluten intolerance" and "gluten-related disorders" are used interchangeably herein. Gluten-related disorders include celiac disease, non-celiac gluten intolerance, gluten intoxication, dermatitis herpetiformis, and wheat allergy.

[0088] Celiac disease Celiac disease is one of the most common immune-mediated disorders. It is found worldwide, particularly in the United States and Europe, where approximately 1% of patients test positive for antibodies. Celiac disease is a complex disorder resulting from the complex interplay between various immunological, genetic, and environmental factors. The 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 irritability, diarrhea, occult stools, steatorrhea, and flatulence.

[0089] Clinical evidence indicates that human leukocyte antigen class II (HLA-DQII), which is strongly associated with celiac disease pathology, 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 crossing 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 site of HLA-DQ2.5 / 8 (Dieterich et al., 1997, Nature medicine, 3(7), 797-801). HLA-DQ2.5 / 8 then presents these specific gluten peptide signals to helper T cells and other immune cells, causing 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).

[0090] Non-celiac gluten sensitivity 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 extraintestinal symptoms induced by gluten ingestion and may improve with the elimination of gluten-containing foods from the diet (Lundin & Alaedini, 2012). The pathogenesis of non-celiac gluten sensitivity remains poorly understood. It has been suggested that proteins / peptides other than gliadin (the major cytotoxic antigen of gluten) present in gluten and gluten-containing grains (wheat, rye, barley, and their derivatives) may be involved in the manifestation of symptoms. Non-celiac gluten sensitivity is the most common syndrome among gluten-related disorders, with 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.

[0091] dermatitis herpetiformis Dermatitis herpetiformis is a chronic autoimmune blistering skin disease characterized by the presence of skin lesions with a widespread, symmetrical distribution, predominating in areas of high friction, primarily affecting both elbows, knees, buttocks, and ankles, but which may also affect the scalp and other parts of the body. The lesions are covered with vesicular crusts, which, when peeled, can lead to localized hyperpigmentation or a chronic, intense burning, itching, and blistering rash.

[0092] The age of onset varies: it can begin in childhood and adolescence, but can affect a subject at any age in their life and in either gender.

[0093] Individuals with dermatitis herpetiformis have varying degrees of intestinal involvement ranging from moderate mucosal involvement to the presence of villous atrophy.

[0094] Wheat allergy The gastrointestinal symptoms of wheat allergy are similar to those of celiac disease and non-celiac gluten sensitivity, but the interval between exposure to wheat and the onset of symptoms differs. Wheat allergy has a rapid onset (minutes to hours) after ingestion of wheat-containing foods and can lead to anaphylaxis.

[0095] Gluten ataxia Gluten ataxia is a gluten-related disorder that causes damage to the cerebellum, the balance center of the brain that controls coordination and complex movements such as walking, speaking, and swallowing. Gluten ataxia is one of the most common causes of sporadic idiopathic ataxia. It accounts for 40% of ataxias of unknown cause and 15% of all ataxias.

[0096] Gluten ataxia is an immune-mediated disease triggered by gluten ingestion in individuals with genetic hypersensitivity. It should be considered in the differential diagnosis of all patients with idiopathic sporadic ataxia. The effectiveness of treatment depends on the time elapsed between the onset of ataxia and diagnosis. Neuronal death in the cerebellum resulting from exposure to gluten in subjects is irreversible.

[0097] Early diagnosis and treatment with a gluten-free diet can improve the condition and prevent its progression. Fewer than 10% of people with gluten ataxia have any gastrointestinal symptoms, but approximately 40% have intestinal damage. Sensitive markers of gluten ataxia include anti-gliadin antibodies. Immunoglobulin A (IgA), which is deposited against transglutaminase 2 (TG2) in the small intestine and extraintestinal sites, has proven more reliable.

[0098] Administration Bifidobacterium longum subsp. longum or the compositions described herein are preferably administered enterally.

[0099] Enteral administration may be oral, gastric, and / or rectal.

[0100] In general terms, administration of the combinations or compositions described herein may be, for example, by the oral route or by another route to the gastrointestinal tract, for example, administration may be by tube feeding.

[0101] In an alternative embodiment, administration of the combinations or compositions described herein may be topical.

[0102] 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.

[0103] Preferred features and embodiments of the present invention will now be described by way of non-limiting examples.

[0104] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology, and immunology which are within the capabilities of those skilled in the art and are explained in the literature. For example, Sambrook, J., Fritsch, EF and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, FM et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley & Sons; Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley&Sons;Polak, JMand McGee, J.O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, MJ (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press and Lilley, DMand. Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures. See Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is incorporated herein by reference.

[0105] [Example] Example 1 - Serpin induction of B. longum CNCM I-2618 (NCC 2705) by galactose B. longum strain CNCM I-2618 (NCC 2705) was grown in a Biolector (growth conditions - anaerobic, 37°C) on a sugar-free MRS + 5mM L-cysteine (MRSc) base supplemented with various carbohydrates.

[0106] Strains were cultured in a Biolector (m2p-labs Aachen, Germany) using 48-well microtiter plates in conjunction with pH and dissolved oxygen (DO) sensors. Plates were continuously shaken for 8 hours to prevent bacterial clumping. 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. The lysate, containing both soluble and insoluble material, was then collected. Total protein content was measured using the PierceBCA kit (Thermofisher), and serpin protein concentration was determined using ELISA.

[0107] As shown in Figure 1, galactose was found to increase B. longum NCC 2705 serpin protein levels compared to all other sugars tested.

[0108] Example 2 - Serpin induction in B. longum CNCM I-2618 (NCC 2705) by galactose in the presence of glucose B. longum NCC 2705 was grown in a sugar-free MRSc medium supplemented with various ratios of glucose and galactose to a final concentration of 1% in a Biolector (as described in Example 1). Cultures were harvested after 18 hours of growth and analyzed for total and serpin protein levels (as described in Example 1).

[0109] The results (Figure 2) show that galactose can induce serpin production in B. longum NCC 2705, even in the presence of glucose, but only if the glucose is present at levels that are depleted during fermentation. In the model system used in this example, a 0.3% addition was the maximum glucose addition rate that could be depleted during fermentation (data not shown). Therefore, it is desirable to keep the glucose concentration of the fermentation system / growth medium low relative to the galactose concentration.

[0110] Example 3 - Serpin induction in B. longum NCC 2705 by galactooligosaccharides (GOS) B. longum NCC 2705 was grown in a sugar-free MRSc medium supplemented with various commercially available galactooligosaccharides (GOS) at various concentrations. Cultures were grown for 18 hours as described previously (see Example 1) and harvested. The resulting pellets were analyzed for total protein and serpin protein content (see Example 1). The commercially available GOS tested were Purimune GOS (from Corn Products International), King GOS (GDS-700-P from King Prebiotics), Vivinal GOS syrup (from DOMO), BMOS (milk oligosaccharides from Nestlé), and Sunfiber® (partially hydrolyzed guar gum from Taiyo GmbH).

[0111] Purimmune GOS, King GOS, Vivinal GOS, and BMOS supported the growth of B. longum NCC 2705. As shown in Figure 3, these GOS were able to significantly increase the levels of serpin proteins in B. longum NCC 2705. Because all commercially available GOS contain residual sugars (mainly glucose and lactose), their concentrations must be adjusted so that the residual sugars are present at levels that are depleted during fermentation. Only Sunfiber R only partially supported the growth of B. longum NCC 2705 (data not shown), but like the other GOS tested, it was able to significantly increase the levels of serpin proteins in B. longum NCC 2705 (Figure 4).

[0112] Example 4 - Serpin induction in B. longum subsp. longum by galactose Serpin-encoding genes and their surroundings are highly conserved within the species B. longum subsp. longum. Strains of B. longum subsp. longum were selected to represent the full range of the genetic tree (Table 2). All strains were cultured in a Biolector (as per Example 1) in a sugar-free MRSc medium supplemented with 1% glucose, 1% galactose, or a mixture of glucose and galactose (0.2% and 0.8%, respectively). Cultures were grown for 18 hours and harvested. The resulting pellets were further analyzed for total protein and serpin protein content (see Example 1).

[0113] [Table 2]

[0114] Not all strains of B. longum subsp. longum were able to grow on galactose as the sole carbohydrate source. Nevertheless, as shown in Figures 5 and 6, it is important to note that serpin protein levels increased in all B. longum subsp. longum strains in the presence of galactose. This indicates that the induction ability of galactose is not dependent on its ability to be metabolized for growth.

[0115] Example 5 - Serpin induction in B. longum by galactose Serpins are also conserved within a limited number of Bifidobacterium species (Turroni, F. et al. Characterization of the serpin-encoding gene of Bifidobacterium breve 210B. Appl Environ Microbiol 76, 3206-3219, doi:10.1128 / AEM.02938-09(2010)). Strains belonging to these species (Table 3) were cultured in a Biolector in a sugar-free MRSc medium supplemented with 1% glucose and 1% galactose (see Example 1). Furthermore, because papain has previously been demonstrated to induce serpins in B. breve, 0.05 mg / mL papain (from Worthington) was also tested in addition to 1% glucose. Cultures were grown for 18 hours and harvested. The resulting pellets were further analyzed for total protein and serpin protein content (see Example 1).

[0116] [Table 3]

[0117] As shown in Figures 5-7, all tested B. longum subsp. longum strains responded to galactose and showed a significant increase in serpin proteins. In contrast, neither B. breve ATCC 15700(T), B. longum subsp. infantis, nor B. longum subsp. suis were induced by galactose. Papain, previously shown to induce serpins in B. breve, did not increase serpin levels in B. longum subsp. longum cultures, but did increase them in B. breve ATCC 15700(T). Two strains belonging to B. longum subsp. infantis and B. longum subsp. suis were not induced by either galactose or papain, respectively (Figure 7).

Claims

1. A medium for increasing serpin protein production in Bifidobacterium longum subsp. longum, comprising galactose, galactose at a concentration of 0.66 to 1.0% by weight and glucose at a concentration that can be depleted during fermentation; A culture medium in which the concentration of residual glucose contained at the end of fermentation of Bifidobacterium longum subsp. longum is 0.02 to 0.3% by weight.

2. A medium for increasing serpin protein production in Bifidobacterium longum subsp. longum, comprising galactooligosaccharides (GOS), GOS at a concentration of 0.25 to 0.9 wt. % and glucose at a concentration that can be depleted during fermentation; A culture medium in which the concentration of residual glucose contained at the end of fermentation of Bifidobacterium longum subsp. longum is 0.02 to 0.3% by weight.

3. 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), ...

3. The medium of claim 1, wherein the medium is selected from the group consisting of Bifidobacterium longum subsp. longum strain ATCC 15707(T), Bifidobacterium longum subsp. I-2170, Bifidobacterium longum subsp. longum strain ATCC 15707(T), and combinations thereof.

4. 1. A method for increasing serpin protein levels in Bifidobacterium longum subsp. longum, said method comprising growing Bifidobacterium longum subsp. longum in a culture medium, said culture medium comprising galactose, GOS, or a combination thereof at a concentration of 0.1 to 1.5% by weight; The method, wherein the residual glucose in the medium is less than 0.4% by weight at the end of fermentation.

5. 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), ... 1-2170, Bifidobacterium longum subsp. longum strain ATCC 15707(T), or a combination thereof.

6. 6. The method of claim 4 or 5, further comprising separating the culture medium after cultivation and / or separating the biomass after cultivation.

7. Bifidobacterium longum subsp. longum prepared by growing it in a medium according to any one of claims 1 to 3.

8. longum strain CNCM I-2169, Bifidobacterium longum subsp. longum strain CNCM I-2171, Bifidobacterium longum subsp. longum strain ATCC BAA-999 (available from Morinaga Milk Industry Co., Ltd. as BB536), 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), ... 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.

9. A composition comprising the cultured biomass described in claim 6, or Bifidobacterium longum subsp. longum described in claim 7 or 8.

10. 10. The composition of claim 9 for use in the treatment or prevention of inflammatory bowel disease, celiac disease, non-celiac gluten sensitivity, gluten intoxication, dermatitis herpetiformis, or wheat allergy.

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