Serine protease inhibitors

Abstract

The present invention discloses the use of galactose or galacto-oligosaccharide for increasing the production of serine protease inhibitor protein in Bifidobacterium longum subspecies longum.

Description

Technical Field

[0001] The present invention relates to bacteria expressing serine protease inhibitors, methods for increasing the production of serine protease inhibitors in bacteria and uses thereof. Background Art

[0002] Gluten-related diseases include all diseases triggered by gluten. They include, among other pathophysiologies, celiac disease and non-celiac gluten sensitivity. Currently, the incidence of a broad spectrum of gluten-related diseases is increasing worldwide, especially in the case of celiac disease and non-celiac gluten sensitivity. Both diseases are triggered by the ingestion of gluten. Celiac disease involves both innate and acquired immunity, while non-celiac gluten sensitivity involves innate immunity.

[0003] A lifelong gluten-free diet is the gold standard therapy for patients with celiac disease and non-celiac gluten sensitivity, but this therapy may have some limitations on the extraintestinal manifestations 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 that following a strict gluten-free diet is very difficult because low-level cross-contamination is difficult to avoid and can occur throughout the food production chain, from grain growing to manufacturing processing (Mitchison et al., 1991, Gut, 32(3), 260-265). In addition, it has been described that up to 3 g of hidden gluten can still be consumed daily on a strict gluten-free diet (Aziz et al., 2014, The American journal of gastroenterology, 109(9), 1498).

[0004] Celiac disease is particularly prevalent in the United States and Europe, where approximately 1% of individuals test positive for antibodies (Dubé et al., 2005, Gastroenterology, 128(4), S57-S67). It is a complex disease caused by a complex interaction between various immune, genetic and environmental factors (Alaedini and Green, 2005). It is triggered by the ingestion of wheat gluten and other related cereal proteins such as rye and barley proteins. Symptoms associated with celiac disease are growth retardation, irritability and delayed puberty in children, as well as a number of gastrointestinal symptoms such as malaise, diarrhea, fecal occult blood, steatorrhea and flatulence (Dubé et al., 2005; Sedghizadeh et al., 2002).

[0005] Non-celiac gluten sensitivity (also known as non-celiac wheat sensitivity) is an emerging disease. It is defined as a clinical entity induced by the intake of gluten, which leads to intestinal symptoms and / or extra-intestinal symptoms that can be improved by removing gluten-containing foods from the diet (Lundin and Alaedini, 2012). In addition to prolamins (the main cytotoxic antigens of gluten), other proteins / peptides present in gluten and gluten-containing cereals (wheat, rye, barley and their derivatives) may play a role in the development of symptoms. Non-celiac gluten sensitivity is the most common syndrome of gluten-related diseases with a prevalence of between 0.5-13% (average 5%) in the general population (Catassi et al., 2013, Nutrients, Vol. 5, No. 10, pp. 3839-3853 (Catassi et al., 2013, Nutrients, 5 (10), 3839-3853)).

[0006] Serpins are a superfamily of proteins present in eukaryotic cells (Gettins, 2002, Chemical reviews, 102(12), 4751-4804) and prokaryotic cells (Kantyka et al., Biochimie, 92(11), 1644-1656).

[0007] Recently, human serine protease inhibitors have been shown to play an important role in gluten-related diseases. Elastin is a human serine protease inhibitor that shows potent inhibitory ability against various forms of elastase and proteases (Ying & Simon, 1993, Biochemistry, vol. 32, no. 7, pp. 1866-1874). Elastin is expressed throughout the epithelium of the gastrointestinal tract, 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, elastin 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 showed that the addition of elastin modestly inhibited transglutaminase 2 (Tg2), thereby inhibiting the deamidation of digestion-resistant 33-mer prolamin peptides, one of the potential triggers of the innate immune response in celiac disease (McCarville et al. 2015, Current opinion in pharmacology, 25, 7-12).

[0008] Delivery of elastin 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 microorganisms (GMOs) and is therefore incompatible with food applications, as consumer acceptance of GMOs is very low.

[0009] Recently, serine protease inhibitors have been reported in prokaryotes. Computer analysis has shown that in different species of bifidobacteria, there are genes encoding serine protease inhibitor-like proteins, especially in the bacteria of Bifidobacterium longum subsp. longum. The protein encoded by Bifidobacterium longum subsp. longum (referred to as B. longum) NCC2705 shows antiprotease activity similar to that of human serine protease inhibitors (Ivanov et al., 2006, Journal of Biological Chemistry, Vol. 281, No. 25, pp. 17246-17252 (Ivanov et al 2006, Journal of Biological Chemistry, 281 (25), 17246-17252)).

[0010] Bifidobacterium longum NCC 2705 was deposited at the Pasteur Institute on January 29, 2001 under the Budapest Treaty with the deposit number CNCM I-2618.

[0011] Bifidobacterium longum NCC 2705 (CNCM I-2618), which has recently been shown to ameliorate gluten-induced pathophysiology in a mouse model of gluten sensitivity by producing serine protease inhibitors, has shown its potential as a solution for gluten-related diseases (McCarville et al., 2017, Appl. Envoron. Microbiol. Vol. 83, no. 19, e01323-17). Summary of the invention

[0012] The present inventors have surprisingly found that galactose and galacto-oligosaccharides (GOS) can increase the production of serine protease inhibitors when added to the bacterial growth medium of Bifidobacterium longum subsp. longum species.

[0013] Thus, in a first aspect of the present invention there is provided the use of galactose or galacto-oligosaccharides (GOS) or a combination thereof for increasing the production of serine protease inhibitors in Bifidobacterium longum subsp. longum.

[0014] In another aspect of the present invention, there is provided a method for increasing the production of a serine protease inhibitor in bacteria of the species Bifidobacterium longum subsp. longum, wherein the method comprises growing Bifidobacterium longum subsp. longum in a culture medium, characterized in that the culture medium comprises galactose or galacto-oligosaccharides (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, characterized in that the culture medium comprises galactose or GOS or a combination thereof.

[0016] The Bifidobacterium longum subsp. longum produced according to the present invention is associated with increased levels of serpin proteins relative 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, Bifidobacterium longum subsp. longum may be cultured in a medium comprising galactose or GOS or a combination thereof at a concentration of, for example, 0.02 to 5 wt %, preferably 0.05 to 2 wt %.

[0018] For example, Bifidobacterium longum strain CNCM 1-2618 can be cultured in a medium comprising 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 produced according to the method described herein.

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

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

[0022] In one embodiment, the composition is a pharmaceutical composition, wherein the pharmaceutical composition 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 produced according to the method described herein, or a composition comprising the Bifidobacterium longum subsp. longum for use in treating or preventing a condition associated with gluten sensitivity or involving reduced activity of a serine protease inhibitor.

[0024] According to another aspect of the present invention, there is provided Bifidobacterium longum subsp. longum produced according to the method described herein, or a composition comprising the Bifidobacterium longum subsp. longum for use in treating or preventing gluten-related diseases.

[0025] According to one aspect of the present invention, there is provided Bifidobacterium longum subsp. longum produced according to the method described herein, or a composition comprising the Bifidobacterium longum subsp. longum subsp. longum for use in treating or preventing 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 produced according to the method described herein, or a composition comprising the Bifidobacterium longum subsp. longum for use in treating or preventing inflammatory bowel disease.

[0027] Bifidobacterium longum subspecies long can be any Bifidobacterium longum subspecies long strain. In some preferred embodiments, the Bifidobacterium longum subspecies long strain can be selected from Bifidobacterium longum subspecies long strain CNCM 1-2169, Bifidobacterium longum subspecies long strain CNCM 1-2171, Bifidobacterium longum subspecies long strain ATCC BAA-999, Bifidobacterium longum subspecies long strain ATCC15708, Bifidobacterium longum subspecies long strain DSM 20097, Bifidobacterium longum subspecies long strain NCIMB 8809, Bifidobacterium longum subspecies long strain CNCM 1-2618 (NCC 2705), Bifidobacterium longum subspecies long strain CNCM 1-2170, Bifidobacterium longum subspecies long strain ATCC 15707 (T), or a combination thereof, particularly Bifidobacterium longum CNCM 1-2618 (NCC 2705).

[0028] It is also understood that galactose and / or GOS may also increase the production of serine protease inhibitors in Bifidobacterium longum subsp. longum in vivo when galactose or GOS or a combination thereof is administered in combination with Bifidobacterium longum subsp. longum.

[0029] Therefore, 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 treating or preventing 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 a combination of Bifidobacterium longum strain CNCM I-2618 and galactose.

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

[0034] In some embodiments, Bifidobacterium longum subspecies long can be selected from Bifidobacterium longum subspecies long strain CNCM 1-2169, Bifidobacterium longum subspecies long strain CNCM 1-2171, Bifidobacterium longum subspecies long strain ATCC BAA-999, Bifidobacterium longum subspecies long strain ATCC 15708, Bifidobacterium longum subspecies long strain DSM 20097, Bifidobacterium longum subspecies long strain NCIMB 8809, Bifidobacterium longum subspecies long strain CNCM 1-2618 (NCC 2705), Bifidobacterium longum subspecies long strain CNCM I-2170, Bifidobacterium longum subspecies long strain ATCC 15707 (T), or a combination thereof.

[0035] In some preferred embodiments, Bifidobacterium longum subspecies long can be selected from Bifidobacterium longum subspecies long strain CNCM I-2169, Bifidobacterium longum subspecies long strain CNCM I-2171, Bifidobacterium longum subspecies long strain ATCC 15708, Bifidobacterium longum subspecies long strain DSM 20097, Bifidobacterium longum subspecies long strain NCIMB 8809, Bifidobacterium longum subspecies long strain CNCM I-2618 (NCC 2705), Bifidobacterium longum subspecies long strain CNCM I-2170, Bifidobacterium longum Bifidobacterium longum strain ATCC 15707 (T) or a combination thereof.

[0036] In some preferred embodiments, Bifidobacterium longum subsp. longum strain Bifidobacterium longum CNCM 1-2618 (NCC 2705) is used. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 - shows the serpin protein levels measured in Bifidobacterium longum NCC 2705 grown for 8 hours on different carbohydrates.

[0038] Figure 2 - shows the serpin protein levels measured in Bifidobacterium longum NCC 2705 grown on different ratios of glucose and galactose.

[0039] Figure 3 - shows the serpin protein levels measured in Bifidobacterium longum NCC 2705 grown on GOS for 8 hours.

[0040] Figure 4 - The effect of (partially hydrolyzed guar gum (PHGG)) on the serine protease inhibitor levels of Bifidobacterium longum NCC 2705 is shown.

[0041] Figure 5 - shows the effect of galactose on the levels of serine protease inhibitors of Bifidobacterium longum subsp. longum strains capable of growing on galactose. Values ​​represent protein levels normalized by the total amount of protein in each sample.

[0042] Figure 6 - shows the effect of galactose on the levels of serine protease inhibitors in a strain of Bifidobacterium longum subsp. longum that is unable to grow on galactose alone. Values ​​represent protein levels normalized by the total amount of protein in each sample.

[0043] Figure 7 - Shown is the effect of galactose, GOS and papain on different Bifidobacterium strains with serine protease inhibitor encoding genes. Values ​​represent protein levels normalized by the total amount of protein in each sample. DETAILED DESCRIPTION

[0044] Composition

[0045] The composition of the invention may be in the form of a food, a medical food, a tube feed, a nutritional composition or a nutritional supplement.The term "nutritional supplement" refers to a product intended to supplement the general diet of an individual.

[0046] In one embodiment, the food product is selected from milk, yoghurt, curd, cheese, fermented milk, fermented milk-based products, rice-based products, milk powder, infant formula and pet food.

[0047] The composition may be in the form of a medical food. As used herein, the term "medical food" refers to a food product specifically formulated for the dietary management of a medical disease or condition. The medical food may be administered under medical supervision. The medical food may be used for oral ingestion or tube feeding.

[0048] The composition may be in the form of a tube feed. The term "tube feed" refers to a product intended to introduce nutrients directly into the gastrointestinal tract of an individual via a feeding tube. Tube feeds may be administered, for example, via a feeding tube placed through the nose of an individual (such as a nasogastric tube, a sinus tube, and a nasojejunal tube) or a feeding tube placed directly into the abdomen of an individual (such as a gastrostomy, gastrojejunostomy, or jejunostomy feeding tube).

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

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

[0051] Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in "Remington's Pharmaceutical Sciences", Mack Publishing Co. (AR 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] The choice of pharmaceutical carrier, excipient or diluent may be selected with reference to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may comprise the following as a carrier, excipient or diluent or in addition to a carrier, excipient or diluent: 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, free-flowing lactose, beta-lactose, 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] Preservatives, stabilizers, dyes and even flavoring agents may be provided in the composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may also be used.

[0056] Nutritionally acceptable carriers, diluents and excipients include those suitable for human or animal consumption as standard in the food industry. Typical nutritionally acceptable carriers, diluents and excipients are familiar to those skilled in the art.

[0057] The composition may be in the form of tablets, dragees, lozenges, capsules, pellets, powders, granules, solutions, emulsions, suspensions, coated granules, spray-dried granules or pills.

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

[0059] It is obvious to those skilled in the art that the ideal dosage will depend on the individual to be treated, its health condition (eg, sex, age or weight) and the route of administration. The ideal dosage used will therefore vary, but can be easily determined by those skilled in the art.

[0060] However, in general, it is preferred that the composition of the present invention contain 10 6 Up to 10 10 cfu and / or 10 6 Up to 10 10 It may also contain 10 per gram of dry weight of the composition. 6 Up to 10 11 cfu and / or 10 6 Up to 10 11 Bifidobacterium longum subsp.

[0061] Bifidobacterium longum

[0062] The Bifidobacterium longum may be any strain of Bifidobacterium longum subsp. longum. In some embodiments, the Bifidobacterium longum subspecies long strain can be selected from Bifidobacterium longum subspecies long strain CNCM I-2169, Bifidobacterium longum subspecies long strain CNCM I-2171, Bifidobacterium longum subspecies long strain ATCC BAA-999 (available as BB536 from Morinaga Milk Industry Co. Ltd), Bifidobacterium longum subspecies long strain ATCC 15708, Bifidobacterium longum subspecies long strain DSM 20097, Bifidobacterium longum subspecies long strain NCIMB 8809, Bifidobacterium longum subspecies long strain CNCM I-2618 (NCC 2705), Bifidobacterium longum subspecies long strain CNCM I-2170, Bifidobacterium longum subspecies long strain ATCC 15707 (T), Bifidobacterium longum subspecies long strain CNCM I-103, Bifidobacterium longum subspecies long strain CNCM I-2334, Bifidobacterium longum subsp. longum strain CNCM I-3864, Bifidobacterium longum subsp. longum strain CNCM I-3853 or a combination thereof.

[0063] The strains have been deposited in the depository institutions indicated in the table below (Table 1) and have been given the following deposit dates and accession numbers:

[0064]

[0065] Table 1

[0066] CNCM refers to the National Collection of Microorganisms, Institut Pasteur, Paris, France (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, USA). DSM refers to the Leibniz Institute for Plant Genetics and Crop Science (Inhoffenstr. 7B, D-38124 Braunschweig, Germany). NCIMB refers to NCIMB Ltd (Ferguson Building, Craibstone Estate, Buckburn, Aberdeen AB21 9YA, Scotland).

[0067] Strains 1, 2, 6, 7, 9, 11-13 have been deposited by Nestlé SA, avenue Nestlé 55, 1800 Vevey, Switzerland. Nestlé SA has since merged into Société des Produits Nestlé S.A. Therefore, Société des Produits Nestlé S.A. is the successor of Nestlé SA under Article 2(ix) of the Budapest Treaty. All other strains are commercially available.

[0068] In some preferred embodiments, Bifidobacterium longum subspecies long can be selected from Bifidobacterium longum subspecies long strain CNCM I-2169, Bifidobacterium longum subspecies long strain CNCM I-2171, Bifidobacterium longum subspecies long strain ATCC 15708, Bifidobacterium longum subspecies long strain DSM 20097, Bifidobacterium longum subspecies long strain NCIMB 8809, Bifidobacterium longum subspecies long strain CNCM I-2618 (NCC 2705), Bifidobacterium longum subspecies long strain CNCM I-2170, Bifidobacterium longum Bifidobacterium longum strain ATCC 15707 (T) or a combination thereof.

[0069] In some preferred embodiments, Bifidobacterium longum subsp. longum strain Bifidobacterium longum CNCM 1-2618 (NCC 2705) is used.

[0070] GOS

[0071] The present inventors have surprisingly found that galactose and galacto-oligosaccharides (GOS) can increase the production of serine protease inhibitors in bacteria of the species Bifidobacterium longum subsp. longum.

[0072] As used herein, the term "oligosaccharide" refers to carbohydrates having a degree of polymerization (DP) ranging from 2 to 20, inclusive.

[0073] "Degree of polymerization" or "DP" refers to the total number of sugar units in an oligosaccharide or polysaccharide chain.

[0074] As used herein, the term "galacto-oligosaccharide" refers to a non-digestible oligosaccharide comprising two or more galactose molecules. The DP of the galacto-oligosaccharide of the present invention is from 2 to 20, preferably from 2 to 10. Based on the monomer subunits, preferably at least 30%, preferably at least 50%, more preferably at least 60% of the sugar units are galactose units.

[0075] Suitable galacto-oligosaccharides are commercially available and include, for example, Purimune GOS (from Corn Products International), King GOS (from King Prebiotics), Vivinal GOS (from Friesland Campina), and PHGG (from Taiyo). Other suppliers 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.

[0076] Due to the configuration of their glycosidic bonds, galacto-oligosaccharides (GOS) are largely resistant to hydrolysis by salivary and intestinal digestive enzymes. GOS are classified as prebiotics, non-digestible carbohydrates that beneficially affect the host by stimulating the growth and / or activity of beneficial bacteria in the colon.

[0077] Bifidobacterium longum subsp. longum can be cultured in a medium comprising galactose or GOS, or a mixture thereof, at a concentration of, for example, 0.02% to 5% by weight. For example, Bifidobacterium longum subsp. longum can be cultured in a medium comprising galactose or GOS, 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.

[0078] Galactose or GOS, or a mixture thereof, may be added to a conventional culture medium containing up to 8 wt%, preferably up to 6 wt%, for example up to 4 wt% of another sugar suitable for maintaining the growth of Bifidobacterium longum, such as but not limited to glucose. The inventors surprisingly found that galactose can induce the production of serine protease inhibitors in Bifidobacterium longum subsp. longum, even in the presence of glucose, but only when glucose is present at a level that allows it to be depleted during fermentation. Preferably, the culture medium at the end of fermentation contains less than 0.4 wt% glucose, such as 0 wt% to 0.3 wt% glucose, for example 0.02 wt% to 0.4 wt%, or about 0.05 wt% to about 0.3 wt%. Conventional culture media suitable for the growth of Bifidobacterium longum are well known to those skilled in the art.

[0079] In one embodiment, Bifidobacterium longum subsp. longum may be cultured in a medium comprising galactose at a concentration of 0.05% to 2%, 0.1% to 1.5% or about 1%, optionally in the presence of glucose at a concentration such that it is depleted until the end of the fermentation. Preferably, the medium at the end of the fermentation contains less than 0.4% glucose, such as 0% to 0.3% glucose. If glucose is present, the medium may contain, for example, 0.02% to 0.4%, or about 0.05% to about 0.3% glucose at the end of the fermentation.

[0080] In one embodiment, Bifidobacterium longum subsp. longum can be cultured in a culture medium comprising GOS at a concentration of 0.05% to 2%, 0.1% to 1.5%, or about 1% by weight, optionally in the presence of residual glucose at a concentration of 0.02% to 0.4%, or about 0.05% to about 0.3% by weight.

[0081] In one embodiment, galactose is used in the above mentioned concentrations.

[0082] In one embodiment, GOS are used in the above concentrations.

[0083] Method for producing culture powder

[0084] Strains belonging to the species Bifidobacterium longum grow under anaerobic conditions. Fermentation methods under anaerobic conditions are well known. Depending on the microorganism to be grown, the skilled person is able to identify suitable components of the fermentation medium and adjust the fermentation conditions based on common sense. The fermentation medium generally comprises

[0085] - a nitrogen source, such as yeast extract;

[0086] - a carbon source, such as sugar;

[0087] - Various growth factors required by microorganisms (such as minerals, vitamins, etc.) and

[0088] -water.

[0089] A non-limiting example of a typical growth medium for Bifidobacterium longum is MRS (De Man, Rogosa and Sharpe) medium (MRSc) supplemented with 0.05% cysteine.

[0090] The fermentation is preferably carried out in two steps, with a starter fermentation being carried out prior to the main fermentation step. The fermentation medium may be different for the starter fermentation and the main fermentation, or may be the same.

[0091] The second step of the method is the concentration of the biomass. This can also be carried out using methods known to those skilled in the art, such as, for example, centrifugation or filtration. Based on the total dry weight of the biomass (i.e. the total amount of the fermentation medium and the microorganism produced), the total solid content of the biomass after concentration is preferably 10% to 35% by weight, preferably 14% to 35% by weight.

[0092] Optionally, concentration can be combined with a washing step before or after the washing step to remove residues of the fermentation medium and / or compounds produced during the fermentation. For example, washing can be performed by concentrating the biomass, resuspending the concentrated biomass in a buffer such as a phosphate buffer or similar composition, and re-concentrating the biomass.

[0093] For example, the methods described in WO 2017 / 001590, which is incorporated by reference in its entirety, may be applied.

[0094] combination

[0095] In 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.

[0096] 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.

[0097] As used herein, the term "simultaneously" or "concurrently" is used to mean that two agents are administered at the same time (ie, at the same time).

[0098] The term "sequentially" or "sequentially" is used to indicate that two agents are administered one after the other, wherein either Bifidobacterium longum subsp. longum, or galactose, or GOS, or a combination thereof, may be administered first.

[0099] The agents may be administered as separate formulations or as a single combined formulation.

[0100] When the compounds are co-formulated, i.e., co-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 common or combined administration of the two compounds.

[0101] In one embodiment, Bifidobacterium longum subsp. longum and galactose or GOS or a combination thereof are in a supplement. In another embodiment, Bifidobacterium longum subsp. longum and galactose or GOS or a combination thereof are in the form of a kit comprising formulations of both agents and optionally instructions for administering the formulations simultaneously or sequentially to a subject in need thereof.

[0102] treat

[0103] The Bifidobacterium longum subsp. longum strain produced according to the present invention or a composition comprising the strain may be used in the treatment or prevention of gluten-related diseases or disorders involving reduced activity of serine protease inhibitors.

[0104] For example, the Bifidobacterium longum subsp. longum produced according to the present invention or a composition comprising the same can be used in the treatment or prevention of inflammatory bowel disease, celiac disease, non-celiac gluten sensitivity, gluten ataxia, dermatitis herpetiformis and wheat allergy.

[0105] Preferably, the disease is a gluten-related disease. Gluten-related diseases encompass diseases triggered by gluten. The terms "conditions associated with gluten sensitivity" and "gluten-related diseases" are used interchangeably herein. Gluten-related diseases include celiac disease, non-celiac gluten sensitivity, gluten ataxia, dermatitis herpetiformis and wheat allergy.

[0106] Celiac disease

[0107] Celiac disease is one of the most common immune-mediated diseases. It is a worldwide condition, especially prevalent in the United States and Europe, where about 1% of individuals test positive for antibodies. Celiac disease is a complex disease caused by a complex interaction between various immune, genetic and environmental factors. It is triggered by the ingestion of wheat gluten and other related grain proteins such as rye and barley proteins. Symptoms associated with celiac disease are growth retardation, irritability and delayed puberty in children, as well as a number of gastrointestinal symptoms such as discomfort, diarrhea, occult blood in stool, steatorrhea and flatulence.

[0108] Clinical evidence shows that class II human leukocyte antigen (HLA-DQII), which is closely related to celiac disease, is expressed in about 95% of celiac disease patients. In the intestinal lumen, gluten is partially digested to form proteolytically resistant 33-mer gluten peptides. After crossing the small intestinal barrier, they are deamidated by negatively charged transglutaminase 2 (TG2) (Sollid, 2000, Annual review of immunology, 18 (1), 53-81), and then they 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, which displays those specific gluten peptides, signals to helper T cells and other immune cells, causing further damage in the small intestine. Antibodies to gluten and autoantibodies to connective tissue components (TG2) are also associated with the progression of celiac disease (Alaedini & Green, 2005, Annals of internal medicine, 142(4), 289-298).

[0109] Non-celiac gluten sensitivity

[0110] Non-celiac gluten sensitivity (also known as non-celiac wheat sensitivity) is an emerging condition. It is defined as a clinical entity induced by the ingestion of gluten, which results in intestinal symptoms and / or extra-intestinal symptoms that can be improved by removing gluten-containing foods from the diet (Lundin and Alaedini, 2012). The pathogenesis of non-celiac gluten sensitivity is not well understood. It has been shown that in addition to prolamins (the main cytotoxic antigen of gluten), other proteins / peptides present in gluten and gluten-containing cereals (wheat, rye, barley and their derivatives) may play a role in the development of symptoms. Non-celiac gluten sensitivity is the most common syndrome of gluten-related diseases with a prevalence of between 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.

[0111] Dermatitis herpetiformis

[0112] Dermatitis herpetiformis is a chronic blistering skin autoimmune disorder characterized by the presence of skin lesions with a widespread and symmetrical distribution, mainly in areas of greater friction, and primarily affecting both elbows, knees, buttocks, ankles, and may also affect the scalp and other parts of the body. These lesions are vesicular-crusting type, and when they flake off, they develop into pigmented areas or a chronic and intense burning, itching and blistering rash.

[0113] The age of onset is variable. It may begin during childhood and adolescence, but may also affect individuals of both sexes indistinctly at any age in their lives.

[0114] People with dermatitis herpetiformis have varying degrees of intestinal lesions, ranging from milder mucosal damage to the presence of villous atrophy.

[0115] Wheat Allergy

[0116] Gastrointestinal symptoms of wheat allergy are similar to those of celiac disease and non-celiac gluten sensitivity, but there are different intervals between exposure to wheat and onset of symptoms. Wheat allergy has a rapid onset (from minutes to hours) after consuming wheat-containing foods and can lead to anaphylaxis.

[0117] Gluten ataxia

[0118] Gluten ataxia is a gluten-related disorder. With gluten ataxia, damage occurs in 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 origin and 15% of all ataxias.

[0119] Gluten ataxia is an immune-mediated disease triggered by the ingestion of gluten in genetically susceptible individuals. It should be considered in the differential diagnosis of all patients with sporadic idiopathic ataxia. The effectiveness of treatment depends on the time elapsed from the onset of ataxia until diagnosis. The death of neurons in the cerebellum caused by exposure of individuals to gluten is irreversible.

[0120] Early diagnosis and treatment with a gluten-free diet can improve ataxia and halt its progression. Fewer than 10% of people with gluten ataxia present with any gastrointestinal symptoms, but about 40% have intestinal damage. Sensitive markers for gluten ataxia include anti-gliadin antibodies. Deposition of immunoglobulin A (IgA) in the small intestine and at extraintestinal sites in close proximity to transglutaminase 2 (TG2) has proven additionally reliable.

[0121] Application

[0122] The Bifidobacterium longum subsp. longum or the composition described herein is preferably administered enterally.

[0123] Enteral administration may be oral, gastric and / or rectal administration.

[0124] Generally, administration of the combinations or compositions described herein may be, for example, by the oral route or another route into the gastrointestinal tract, such as by tube feeding.

[0125] In an alternative embodiment, administration of the combinations or compositions described herein may be topical.

[0126] The subject may be a mammal, such as a human, canine, feline, equine, caprine, bovine, ovine, porcine, cervidae, and primate. Preferably, the subject is a human.

[0127] Preferred features and embodiments of the invention will now be described by way of non-limiting examples.

[0128] 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 one of ordinary skill in the art and are described in the literature. See, e.g., Sambrook, J., Fritsch, EF, and Maniatis, T., 1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press; Ausubel, FM et al., (1995 and periodic supplements), Current Protocols in Molecular Biology, Chapters 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. These general texts are incorporated herein by reference.

[0129] Example

[0130] Example 1: Inhibition of Bifidobacterium longum CNCM 1-2618 (NCC 2705) serine protease by galactose Induced by

[0131] Bifidobacterium longum strain CNCM 1-2618 (NCC 2705) was grown in a sugar-free MRS + 5 mM L-cysteine ​​(MRSc) base in a microbioreactor (Biolector) (growth conditions—anaerobic, 37° C.) to which different carbohydrates were added.

[0132] The strains were cultured in a microbioreactor (m2p-labs Aachen, Germany) using a 48-well microtiter plate equipped with a pH sensor and a dissolved oxygen (DO) sensor. The strains were shaken continuously for 8 hours to prevent bacterial aggregation. The culture was harvested by centrifugation and the supernatant was removed. The precipitate was resuspended in PBS supplemented with a halt protease inhibitor (Sigma) and lysed using glass beads. The lysate containing soluble and insoluble materials was then collected. The total protein content was measured using a Pierce BCA kit (Thermofisher), and the serine protease inhibitor protein concentration was determined using ELISA.

[0133] like Figure 1 As shown, galactose was shown to increase Bifidobacterium longum NCC2705 serpin protein levels compared to all other sugars tested.

[0134] Example 2: Isolation of Bifidobacterium longum CNCM I-2618 (NCC 2705) filaments by galactose in the presence of glucose PI-induced

[0135] Bifidobacterium longum NCC 2705 was cultured in a microbioreactor in a sugar-free MRSc base (as described in Example 1) with different ratios of glucose and galactose added to a final concentration of 1%. Cultures were harvested after 18 hours of growth and analyzed for total serpin protein levels (as described in Example 1).

[0136] result( Figure 2 ) showed that galactose can induce the production of serine protease inhibitors in Bifidobacterium longum NCC 2705, even in the presence of glucose, but only when glucose is present at a level that is depleted during fermentation. In the model system used in this example, the addition of 0.3% is the maximum addition rate of glucose, allowing it to be depleted during fermentation (data not shown). Therefore, the glucose concentration in the fermentation system / growth medium should be kept low relative to the galactose concentration.

[0137] Example 3: Induction of Serine Protease Inhibitors of Bifidobacterium longum NCC 2705 by Galacto-oligosaccharides (GOS) guide

[0138] Bifidobacterium longum NCC 2705 is grown on the MRSc base without sugar, and different commercially available galacto-oligosaccharides (GOS) are added at different concentrations. Culture is grown for 18 hours and gathered in the crops as previously described (referring to Example 1). The total protein content and the serine protease inhibitor protein content (referring to Example 1) of the precipitation obtained by analysis. The commercial GOS tested is Purimune GOS (derived from International Corn Products Company), King GOS (GDS-700-P, derived from Jinou Liduo Company), Vivinal GOS syrup (derived from DOMO Company (DOMO)), BMOS (cow's milk oligosaccharide, derived from Nestle Company (Nestlé)), Sunfiber R (partially hydrolyzed guar gum; Derived from derived from Sun Company (Taiyo GmbH)).

[0139] Purimmune GOS, King GOS, Vivinal GOS, and BMOS supported the growth of Bifidobacterium longum NCC 2705. Figure 3 As shown, these GOS can significantly increase the level of serpin protein in Bifidobacterium longum NCC 2705. Since commercially available GOS all contain residual sugars (mainly glucose and lactose), the concentration used should be adjusted so that those residual sugars are present at a level that is depleted during fermentation. Sunfiber R alone only partially supported the growth of Bifidobacterium longum NCC 2705 (data not shown), however, like the other GOS tested, it was able to significantly increase the level of serpin protein in Bifidobacterium longum NCC 2705 ( Figure 4 ).

[0140] Example 4: Induction of Bifidobacterium longum subsp. longum Serine Protease Inhibitor by Galactose

[0141] The serine protease inhibitor encoding gene and its surroundings are highly conserved in the long subspecies of Bifidobacterium longum. The strains of long subspecies of Bifidobacterium longum were selected to represent the entire span of the genetic phylogenetic tree (Table 2). All strains were cultured in a sugar-free MRSc base in a microbioreactor (according to Example 1), to which a mixture of 1% glucose, 1% galactose or glucose and galactose (0.2% and 0.8% respectively) was added. The cultures were grown for 18 hours and harvested. The total protein content and serine protease inhibitor protein content of the precipitates obtained were further analyzed (see Example 1).

[0142] Table 2: List of tested B. longum subsp. longum strains and homology of their serpin genes to BL0108 (B. longum NCC 2705 serpin encoding gene).

[0143] strain n° %ID of BL0108 (NCC2705 serine protease inhibitor) NCC 2705(CNCM I-2618) 100.00 ATCC 15707(T) 99.78 CNCN I-2171 99.79 ATCC BAA-999 99.78 ATCC 15708 99.57 DSM 20097 97.42 NCIMB 8809 99.79 CNCM I-2170 100

[0144] All strains of Bifidobacterium longum subsp. longum were able to grow on galactose as a sole carbohydrate source. Figure 5 and Figure 6 As shown in , importantly, serpin protein levels increased in all B. longum subsp. longum strains in the presence of galactose, implying that the inducible capacity of galactose is independent of its ability to metabolize growth.

[0145] Example 5: Induction of Bifidobacterium longum Serine Protease Inhibitor by Galactose In addition, serine protease inhibitors are conserved in 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 sugar-free MRSc base in a microbioreactor (see Example 1), to which 1% glucose, 1% galactose were added. Likewise, 0.05 mg / ml papain (obtained from Worthington) was tested on top of 1% glucose, as previously demonstrated to induce serine protease inhibitors in B. breve. The cultures were grown for 18 hours and harvested. The pellets obtained were further analyzed for total protein content and serpin protein content (see Example 1).

[0146] Table 3: List of strains used and the homology of their serine protease inhibitor genes to BL0108 (Bifidobacterium longum NCC 2705 serine protease inhibitor encoding gene).

[0147]

[0148] like Figure 5-Figure 7As shown, all tested B. longum subsp. longum strains responded to galactose and showed a significant increase in serpin proteins. In contrast, B. breve ATCC 15700 (T), B. longum subsp. infantis and B. longum subsp. suis strains were not induced by galactose. Papain, which was previously shown to induce serpins in B. longum subsp. longum cultures, did not increase serpin levels in B. longum subsp. longum cultures, but did so in B. breve ATCC 15700 (T) strains. Two strains belonging to B. longum subsp. infantis and B. longum subsp. suis, respectively, were neither induced by galactose nor by papain ( Figure 7 ).

Claims

1. Use of galactose, galacto-oligosaccharides (GOS) or a combination thereof for increasing the production of a serine protease inhibitor protein in Bifidobacterium longum subsp. longum, wherein the culture medium comprises galactose, GOS or a combination thereof at a concentration of 0.02% to 2% by weight, and in the presence of glucose at a concentration that enables it to be consumed before the end of fermentation.

2. The use according to claim 1, wherein the culture medium comprises GOS at a concentration of 0.02% to 2% by weight, and residual glucose at a concentration of 0.02% to 0.3% by weight.

3. The use according to any one of claims 1 to 2, wherein the Bifidobacterium longum subspecies long is selected from Bifidobacterium longum subspecies long strain CNCM 1-2169, Bifidobacterium longum subspecies long strain CNCM 1-2171, Bifidobacterium longum subspecies long strain ATCC BAA-999, Bifidobacterium longum subspecies long strain ATCC 15708, Bifidobacterium longum subspecies long strain DSM 20097, Bifidobacterium longum subspecies long strain NCIMB 8809, Bifidobacterium longum subspecies long strain CNCM 1-2618, Bifidobacterium longum subspecies long strain CNCM 1-2170, Bifidobacterium longum subspecies long strain ATCC 15707, or a combination thereof.

4. A method for increasing the level of a serpin protein in Bifidobacterium longum subsp. longum, wherein the method comprises growing Bifidobacterium longum subsp. longum in a culture medium, characterized in that The medium comprises galactose or galacto-oligosaccharides (GOS) or a combination thereof at a concentration ranging from 0.02% to 2% by weight, and in the presence of glucose at a concentration that enables it to be consumed before the end of the fermentation.

5. The method of claim 4, wherein the culture medium comprises GOS at a concentration of 0.02 to 2 wt%, and residual glucose at a concentration of 0.02 to 0.3 wt%.

6. The method according to claim 4 or 5, wherein the Bifidobacterium longum subspecies long is selected from the group consisting of Bifidobacterium longum subspecies long strain CNCM 1-2169, Bifidobacterium longum subspecies long strain CNCM 1-2171, Bifidobacterium longum subspecies long strain ATCC BAA-999, Bifidobacterium longum subspecies long strain ATCC 15708, Bifidobacterium longum subspecies long strain DSM 20097, Bifidobacterium longum subspecies long strain NCIMB 8809, Bifidobacterium longum subspecies long strain CNCM 1-2618, Bifidobacterium longum subspecies long strain CNCM I-2170, Bifidobacterium longum subspecies long strain ATCC 15707, or a combination thereof.

7. Bifidobacterium longum subspecies longum produced by a method of growing Bifidobacterium longum subspecies longum in a culture medium, characterized in that The medium comprises galactose or GOS or a combination thereof at a concentration ranging from 0.02% to 2% by weight, and in the presence of glucose at a concentration that enables it to be consumed before the end of the fermentation.

8. Bifidobacterium longum subsp. longum according to claim 7, wherein the culture medium comprises GOS at a concentration of 0.02 to 2 wt%, and residual glucose at a concentration of 0.02 to 0.3 wt%.

9. The Bifidobacterium longum subspecies according to claim 7 or 8, wherein the Bifidobacterium longum subspecies is selected from the group consisting of Bifidobacterium longum subspecies CNCM I-2169, Bifidobacterium longum subspecies CNCM I-2171, Bifidobacterium longum subspecies ATCC BAA-999, Bifidobacterium longum subspecies ATCC 15708, Bifidobacterium longum subspecies DSM20097, Bifidobacterium longum subspecies NCIMB 8809, Bifidobacterium longum subspecies CNCM I-2618, Bifidobacterium longum subspecies CNCM I-2170, Bifidobacterium longum subspecies ATCC 15707, Bifidobacterium longum subspecies CNCM I-103, Bifidobacterium longum subspecies CNCM I-2334, Bifidobacterium longum subspecies CNCM I-3864, Bifidobacterium longum subspecies CNCM I-3853, or a combination thereof.

10. Bifidobacterium longum subsp. longum according to any one of claims 7 to 9, for use in the treatment or prevention of inflammatory bowel disease, celiac disease, non-celiac gluten sensitivity, gluten ataxia, dermatitis herpetiformis or wheat allergy.

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