Serine protease inhibitor production

Abstract

The present invention provides the use of a sugar selected from lactose, fructose and raffinose for increasing the expression of a serine protease inhibitor in the Bifidobacterium longum strain CNCM I-2618.

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 disorders 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 disorders 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, Gastroenterology, vol. 32, no. 3, pp. 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, vol. 109, no. 9, pp. 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 disorder 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 gluten. 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 disorders with a prevalence of between 0.5% and 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 disorders. Elastin is a human serine protease inhibitor that shows potent inhibitory ability against various forms of elastase and proteases (Ying and 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, and 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 joumal 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. In silico analysis has shown the presence of genes encoding serine protease inhibitor-like proteins in different Bifidobacterium species. The protein encoded by B. longum subsp longum (referred to as B. longum) NCC 2705 showed 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).

[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 disorders (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 specific sugars can increase the expression of serine protease inhibitors when added to the growth medium of Bifidobacterium longum strain CNCM 1-2618 (B. longum NCC2705).

[0013] Therefore, in a first aspect of the present invention, there is provided the use of a sugar selected from lactose, fructose and raffinose or a combination thereof for increasing the expression of a serine protease inhibitor in the Bifidobacterium longum strain CNCM 1-2618.

[0014] In another aspect of the present invention, there is provided a method for increasing the expression of a serine protease inhibitor in the Bifidobacterium longum strain CNCM I-2618, wherein the method comprises growing the Bifidobacterium longum strain CNCM I-2618 in a culture medium, characterized in that the culture medium comprises a sugar selected from lactose, fructose and raffinose or a combination thereof.

[0015] According to another aspect of the present invention, there is provided Bifidobacterium longum strain CNCM I-2618 produced by a method of growing Bifidobacterium longum strain CNCM I-2618 in a culture medium, characterized in that the culture medium contains a sugar selected from lactose, fructose and raffinose or a combination thereof.

[0016] The Bifidobacterium longum strain CNCM 1-2618 according to the invention is associated with a higher production of serine protease inhibitors relative to the Bifidobacterium longum strain CNCM 1-2618 grown in the absence of lactose, fructose or raffinose or a combination thereof.

[0017] In one embodiment, the sugar is lactose.

[0018] According to the invention, Bifidobacterium longum strain CNCM 1-2618 may be cultivated in a medium comprising a sugar selected from lactose, fructose or raffinose or a combination thereof in a concentration of, for example, 0.02% to 0.50% by weight.

[0019] For example, Bifidobacterium longum strain CNCM 1-2618 may be cultured in a medium comprising a sugar selected from lactose, fructose or raffinose or a combination thereof at a concentration of 0.05 to 0.15 wt%, 0.08 to 0.12 wt% or about 0.10 wt%.

[0020] According to another aspect of the present invention, there is provided a composition comprising the Bifidobacterium longum strain CNCM 1-2618 produced according to the method described herein.

[0021] In one embodiment, the composition is a food, a medical food, a tube feed, or a nutritional supplement.

[0022] 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.

[0023] In one embodiment, the composition is a pharmaceutical composition, wherein the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0024] According to another aspect of the present invention, there is provided a Bifidobacterium longum strain CNCM 1-2618 produced according to the method described herein, or a composition comprising the Bifidobacterium longum strain CNCM 1-2618, for use in treating or preventing conditions associated with gluten sensitivity or involving reduced activity of serine protease inhibitors.

[0025] According to another aspect of the present invention, there is provided Bifidobacterium longum strain CNCM I-2618 produced according to the method described herein, or a composition comprising the Bifidobacterium longum strain CNCM I-2618, for use in treating or preventing gluten-related disorders.

[0026] According to one aspect of the present invention, there is provided a Bifidobacterium longum strain CNCM I-2618 produced according to the method described herein, or a composition comprising the Bifidobacterium longum strain CNCM I-2618, for use in treating or preventing celiac disease, non-celiac gluten sensitivity, gluten ataxia, dermatitis herpetiformis or wheat allergy.

[0027] According to another aspect of the present invention, there is provided Bifidobacterium longum strain CNCM I-2618 produced according to the method described herein, or a composition comprising the Bifidobacterium longum strain CNCM I-2618, for use in treating or preventing inflammatory bowel disease.

[0028] It will also be appreciated that the saccharides may also increase the production of serine protease inhibitors in the Bifidobacterium longum strain CNCM 1-2618 in vivo when the saccharides are administered in combination with the Bifidobacterium longum strain CNCM 1-2618.

[0029] Therefore, according to another aspect of the present invention, there is also provided a combination of (i) Bifidobacterium longum strain CNCM 1-2618 and (ii) a sugar selected from lactose, fructose and raffinose or a combination thereof.

[0030] According to another aspect of the present invention, there is also provided a combination of (i) Bifidobacterium longum strain CNCM 1-2618 and (ii) a sugar selected from lactose, fructose and raffinose or a combination thereof for use in treating or preventing a condition associated with gluten sensitivity or a condition associated with a reduced level of a serine protease inhibitor.

[0031] In one embodiment, the combination is a combination of Bifidobacterium longum strain CNCM 1-2618 and lactose.

[0032] According to another aspect of the present invention, there is also provided the Bifidobacterium longum strain CNCM 1-2618 for use in the treatment or prevention of conditions associated with gluten sensitivity or conditions associated with reduced levels of serine protease inhibitors, wherein the Bifidobacterium longum strain CNCM 1-2618 is administered in combination with a sugar selected from lactose, fructose and raffinose or a combination thereof.

[0033] According to another aspect of the invention, there is provided a sugar selected from lactose, fructose and raffinose or a combination thereof for use in treating or preventing a condition associated with gluten sensitivity or a condition associated with a reduced level of a serine protease inhibitor, wherein the sugar is administered in combination with Bifidobacterium longum strain CNCM 1-2618. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 - Relative transcript levels of serpin genes in Bifidobacterium longum NCC 2705 after 180 minutes of sugar induction at a concentration of 0.1%. The bars indicate the relative amount of serpin mRNA for a particular sample compared to growth in MRSc alone. Black bars represent significant induction. Standard deviations were obtained from at least 2 different experiments (95% confidence intervals).

[0035] Figure 2 - Serine protease inhibitor mRNA levels of Bifidobacterium longum NCC2705 fermented in different growth media after 8 hours, 12 hours and 16 hours. Different proportions of glucose are replaced by lactose and fructose, as shown on the y-axis, and the total sugar percentage remains stable at 5.5%. These values ​​are calculated based on the growth of Bifidobacterium longum NCC2705 in 5.5% glucose after 8 hours. Black bars represent significant induction. DETAILED DESCRIPTION

[0036] Composition

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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).

[0041] 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.

[0042] Examples of such suitable excipients for use in the compositions described herein may be found in "Handbook of Pharmaceutical Excipients", 2nd Edition, (1994), Edited by A Wade and PJ Weller.

[0043] 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).

[0044] Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, etc. Examples of suitable diluents include ethanol, glycerol and water.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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 10 cells of Bifidobacterium longum strain CM I-2618. 6 Up to 10 11 cfu and / or 10 6 Up to 10 11 Bifidobacterium longum strain CM I-2618 cells.

[0053] sugar

[0054] Sugars for use in the present invention are selected from lactose, fructose and raffinose or combinations thereof. Lactose is a disaccharide present in milk and composed of glucose and galactose. Fructose is a monosaccharide present in many plants. Raffinose is a trisaccharide also present in plants and composed of galactose, glucose and fructose.

[0055] Bifidobacterium longum strain CNCM I-2618 can be cultured in a medium comprising lactose, fructose or raffinose or a mixture thereof at a concentration of, for example, 0.02% to 0.50% by weight. For example, Bifidobacterium longum strain CNCM I-2618 can be cultured in a medium comprising lactose, fructose or raffinose or a mixture thereof at a concentration of 0.05% to 0.15%, 0.08% to 0.12% or about 0.10% by weight.

[0056] Lactose, fructose, raffinose or mixtures thereof may be added to a conventional medium containing up to 8 wt%, preferably up to 6 wt%, for example 4 wt% to 6 wt% of another sugar suitable for maintaining the growth of Bifidobacterium longum, such as but not limited to glucose or sucrose. Conventional medium suitable for the growth of Bifidobacterium longum is well known to those skilled in the art.

[0057] In one embodiment, the Bifidobacterium longum strain CNCM 1-2618 may be cultured in a medium comprising sugars at a concentration of 0.03 to 0.40 wt%, 0.04 to 0.30 wt%, or 0.05 to 0.20 wt%.

[0058] In one embodiment, the culture medium may contain lactose, fructose, or raffinose, or a combination thereof, at a concentration of 0.03% to 0.15%, 0.04% to 0.15%, 0.05% to 0.15%, 0.06% to 0.15%, 0.07% to 0.15%, 0.08% to 0.15%, 0.09% to 0.15%, or 0.10% to 0.15%.

[0059] In one embodiment, the culture medium may comprise lactose, fructose, or raffinose, or a combination thereof, at a concentration of 0.05% to 0.14%, 0.05% to 0.13%, 0.05% to 0.12%, or 0.05% to 0.11% by weight.

[0060] In one embodiment, the culture medium may contain lactose, fructose, or raffinose, or a combination thereof, at a concentration of 0.06% to 0.14%, 0.07% to 0.13%, 0.08% to 0.12%, 0.09% to 0.11%, or about 0.10% by weight.

[0061] In one embodiment, lactose is used in the above mentioned concentrations.

[0062] In one embodiment, fructose is used in the above-mentioned concentrations.

[0063] In one embodiment, raffinose is used in the above concentrations.

[0064] Method for producing culture powder

[0065] 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

[0066] - a nitrogen source, such as yeast extract;

[0067] - a carbon source, such as sugar;

[0068] - Various growth factors required by microorganisms (such as minerals, vitamins, etc.) and

[0069] -water.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] For example, the methods described in WO 2017 / 001590, which is incorporated by reference in its entirety, may be applied.

[0075] combination

[0076] In one aspect of the present invention, there is provided a combination of (i) Bifidobacterium longum strain CNCM 1-2618 and (ii) a sugar selected from the group consisting of lactose, fructose and raffinose.

[0077] As used herein, the term "combination" refers to the combined administration of Bifidobacterium longum strain CNCM 1-2618 and lactose, fructose or raffinose, wherein the Bifidobacterium longum strain CNCM 1-2618 and the sugar may be administered simultaneously or sequentially.

[0078] As used herein, the term "simultaneously" or "concurrently" is used to indicate that the two agents (Bifidobacterium longum strain CNCM 1-2618 and the sugar) are administered simultaneously (ie at the same time).

[0079] The term "sequentially" or "sequentially" is used to indicate that the two agents are administered one after the other, wherein the Bifidobacterium longum strain CNCM 1-2618 may be administered first, or the sugar may be administered first.

[0080] The agents may be administered as separate formulations or as a single combined formulation.

[0081] 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 agents in the same formulation or separate formulations can also be described as the common or combined administration of the two compounds.

[0082] In one embodiment, the Bifidobacterium longum strain CNCM 1-2618 and the sugar are mixed. In another embodiment, the Bifidobacterium longum strain CNCM 1-2618 and the sugar are present in the form of a kit containing a preparation of the two agents and instructions for administering the preparation simultaneously or sequentially to an individual in need thereof.

[0083] treat

[0084] The Bifidobacterium longum strain CNCM 1-2618 produced according to the present invention or a composition comprising the strain can be used for the treatment or prevention of gluten-related disorders or conditions involving reduced activity of serine protease inhibitors.

[0085] For example, the Bifidobacterium longum strain CNCM 1-2618 produced according to the present invention or a composition comprising the strain can be used to treat or prevent inflammatory bowel disease, celiac disease, non-celiac gluten sensitivity, gluten ataxia, dermatitis herpetiformis and wheat allergy.

[0086] Preferably, the disease is a gluten-related disorder. Gluten-related disorders encompass diseases triggered by gluten. The terms "conditions associated with gluten sensitivity" and "gluten-related disorders" are used interchangeably herein. Gluten-related disorders include celiac disease, non-celiac gluten sensitivity, gluten ataxia, dermatitis herpetiformis and wheat allergy.

[0087] Celiac disease

[0088] Celiac disease is one of the most common immune-mediated disorders. 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 disorder 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.

[0089] 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) have also been associated with the progression of celiac disease (Alaedini & Green, 2005, Annals of internal medicine, 142(4), 289-298).

[0090] Non-celiac gluten sensitivity

[0091] 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 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). The pathogenesis of non-celiac gluten sensitivity is not yet fully understood. It has been shown that 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 disorders with a prevalence of between 0.5% and 13% in the general population (Catassi et al., 2013, Nutrients, Vol. 5, No. 10, pp. 3839-385). The diagnosis of non-celiac gluten sensitivity is made by excluding other gluten-related disorders.

[0092] Dermatitis herpetiformis

[0093] 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.

[0094] 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.

[0095] People with dermatitis herpetiformis have varying degrees of intestinal lesions, ranging from milder mucosal damage to the presence of villous atrophy.

[0096] Wheat Allergy

[0097] The 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 result in anaphylaxis.

[0098] Gluten ataxia

[0099] 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.

[0100] 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. Neuronal death in the cerebellum caused by exposure of individuals to gluten is irreversible.

[0101] 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.

[0102] Application

[0103] The Bifidobacterium longum strain CNCM 1-2618 or the composition described herein is preferably administered enterally.

[0104] Enteral administration may be oral, gastric and / or rectal administration.

[0105] 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.

[0106] In an alternative embodiment, administration of the combinations or compositions described herein may be topical.

[0107] 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.

[0108] Preferred features and embodiments of the invention will now be described by way of non-limiting examples.

[0109] 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, for example, Sambrook, J., Fritsch, EF and Maniatis, T. (1989) Molecular Cloning: ALaboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press; Ausubel, FM et al (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch.9, 13and 16, John Wiley&Sons; Roe, B., Crabtree, J. and Kahn, A., (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley&Sons; Polak, JM and 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, DM And Dahlberg, JE (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is incorporated herein by reference.

[0110] Example

[0111] Example 1 - Method

[0112] BL NCC 2705 (Bifidobacterium longum strain CNCM 1-2618) was grown in a Biolector (growth conditions—anaerobic, 37° C., MRS + 5 mM L-cysteine ​​(MRSc)).

[0113] The strains were cultured in Biolector (m2p-labs Aachen, Germany) using a 48-well microtiter plate equipped with a pH sensor and a dissolved oxygen (DO) sensor. Continuous shaking was used to prevent bacterial aggregation and measurements were taken every 15 minutes. The plates were sealed with a breathable membrane and the anaerobic chamber provided clear anaerobic degassing conditions. Fermentation parameters such as biomass, pH and dissolved oxygen (DO) were monitored online using BioLection HMI software throughout the growth process. Growth parameters were:

[0114] Vaccination rate (%)2

[0115] Volume of each wall (ml)1

[0116] Fermentation time (h) 16

[0117] Temperature(℃)37

[0118] External gas supplied N 2

[0119] External gas pressure (bar)2

[0120] Shaking frequency (rpm) 400

[0121] Measurement cycle time (min) 15

[0122] The following carbohydrates were added to the cultures individually:

[0123] Monosaccharides - glucose, N-acetyl-glucosamine, fructose, galactose.

[0124] Disaccharides - lactose, melibiose, sucrose

[0125] Polysaccharide-raffinose,

[0126] All sugars were purchased from Sigma-Aldrich.

[0127] Bifidobacterium longum NCC 2705 was then further cultured under the same conditions for 180 minutes, after which samples (500 μl) were collected and the serpin mRNA levels were measured. Each fermentation was performed at least twice.

[0128] like Figure 1 As shown, lactose, fructose and raffinose were shown to increase the level of serpin mRNA in Bifidobacterium longum NCC 2705.

[0129] Serine protease inhibitor mRNA detection and quantification

[0130] Total RNA Isolation - 500 μl of bacterial culture was suspended in 1 mL of RNA Protect Bacteria Reagent (Qiagen, Germany) and briefly centrifuged at 5000 g for 10 min to harvest cells for total RNA extraction.

[0131] Total RNA was extracted using the RNeasy total RNA mini kit (Qiagen, Germany, product number 74101) with proteinase K (Qiagen, Germany, product number 19131) and further processed using the RNase-free DNase set (Qiagen, Germany, product number 79254). Total RNA was then eluted in 40 μl of water.

[0132] RNA quality control and quantification - Quality control and quantification of RNA samples were analyzed by electrophoresis using the QIAxcel Advanced system (Qiagen, Germany).

[0133] 1 μl RNA sample and RNA size marker were mixed with the same volume of RNA denaturation buffer, incubated at 70°C for 2 minutes, and then cooled on ice for 1 minute. The sample was diluted to 10 μl by QX RNA diluent for analysis and measurement. The ratio of 23S to 16S was used to assess RNA quality. RNA with a ratio value between 1.6 and 2.3 was selected for subsequent qRT-PCR.

[0134] Both cDNA synthesis and PCR were performed in a one-step reaction using the ABI Prism 7900HT system equipped with the SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit with ROX (Invitrogen, Cat. No. 11746-500).

[0135] PCR products were detected with SYBR green fluorescent dye and normalized with ROX reference dye.

[0136] The following primers were used for cDNA synthesis: for the serpin gene: forward 5'-ACCAATCGCTGCTAAGTTCG-3', reverse 5'-TCGCTGGCAAGAGAGTAGTC-3'; for ldh: forward 5'-CGAACGCCATCTACATGCTC-3' and reverse 5'-AAGATCTGGTTCTCTTGCAG-3'. Primers for serpin were created based on Bifidobacterium longum NCC2705, and DNA homology was checked in Bifidobacterium longum ATCC15707. Reliability was verified by dissociation curve analysis.

[0137] mRNA fold expression analysis method - Relative transcriptional changes were calculated using the Pfaffl method (Pfaffl, 2012, Martin Filion, Hg., Quantitative real-time PCR in Applied Microbiology, 53-62), the formula of which is shown in Equation 1:

[0138] Formula 1

[0139]

[0140] Example 2 - Results

[0141] In order to optimize the effect of lactose on the induction of serpins in BL NCC 2705, a dose response was performed. Fermentation was performed in a 10 L Biostar B Plus fermentor filled with MRSc medium supplemented with 0.05% cysteine ​​as a base, and different concentrations of lactose (0.05%, 0.1%, 1%, 2% and 2.75%) were added to the medium. Glucose was supplemented to the different recipes to reach a total amount of 5.5% of the final total sugar. Serpin mRNA induction folds were calculated using 8 h MRSc grown BL NCC2705 cultures as a base ( Figure 2 ).

[0142] like Figure 2 As shown, the highest serpin mRNA level reached at the end of fermentation (16 h) by using lactose was observed with the addition of 0.1% lactose.

[0143]

[0144] For receiving office use only

[0145]

[0146] For international patent office use only

[0147]

Claims

1. Use of a sugar selected from the group consisting of lactose, fructose and raffinose for increasing the expression of a serine protease inhibitor in the Bifidobacterium longum strain CNCM I-2618, wherein the Bifidobacterium longum strain CNCM I-2618 is cultured in a medium comprising the sugar selected from the group consisting of lactose, fructose and raffinose at a concentration of 0.05% to 0.1% by weight.

2. The use according to claim 1, wherein the culture medium comprises the sugar selected from the group consisting of lactose, fructose and raffinose in a concentration of 0.08% to 0.1% by weight.

3. The use according to claim 1, wherein the culture medium comprises the sugar selected from the group consisting of lactose, fructose and raffinose at a concentration of 0.1% by weight.

4. Use according to any one of claims 1 to 3, wherein the sugar is lactose.

5. A method for increasing the expression of a serine protease inhibitor in the Bifidobacterium longum strain CNCM I-2618, wherein the method comprises growing the Bifidobacterium longum strain CNCM I-2618 in a culture medium, characterized in that The culture medium comprises a sugar selected from the group consisting of lactose, fructose and raffinose, wherein the culture medium comprises the sugar at a concentration of 0.05 wt % to 0.1 wt %.

6. The method according to claim 5, wherein the culture medium comprises the sugar at a concentration of 0.08 wt% to 0.1 wt%.

7. The method according to claim 5, wherein the culture medium comprises the sugar at a concentration of 0.1 wt%.

8. The method according to any one of claims 5 to 7, wherein the sugar is lactose.

Images