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  • 专利说明
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    • 专利摘要:
    The invention relates to a strain selected from Lactobacillus rhamnosus L8.6, Lactobacillus rhamnosus N4.7 and Bifidobacterium breve N24.5 or a combination thereof and compositions comprising them, for use in the treatment of disorders related to the ingestion of gluten, like celiac disease. The strains can be used in pharmaceutical compositions, in functional foods, probiotic foods, symbiotic foods, food supplements or nutraceuticals. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2698566A1
    申请号:ES201830518
    申请日:2018-05-31
    公开日:2019-02-05
    发明作者:Blanco Francisco Javier Casqueiro；Andrés Jenifer Pérez；Blázquez Cristina Iglesias；Ruiz De Morales José María García；Aparicio Leandro Benito Rodriguez；García Miguel Angel Ferrero
    申请人:Gerencia Regional De Salud De Castilla Y Leon；Universidad de Leon；
    IPC主号:
    专利说明:

    [0001] MICROBIAL STATES, PHARMACEUTICAL COMPOSITIONS AND FOODS THAT CONTAIN THEM FOR THE TREATMENT OF RELATED DISORDERS
    [0002]
    [0003] TECHNICAL SECTOR
    [0004]
    [0005] The present invention belongs to the fields of the food industry and the pharmaceutical industry, more specifically to food or pharmaceutical products comprising microorganisms involved in the metabolism of gluten.
    [0006]
    [0007] BACKGROUND OF THE INVENTION
    [0008]
    [0009] Celiac disease or celiac disease is a chronic inflammatory bowel disease mediated by the immune system. The disease produces lesions in the epithelium and in the lamina propria of the small intestine such as villous atrophy, hyperplasia in the crypts and leukocyte infiltration. Clinically it presents a great variety of symptoms, both gastrointestinal and extra-intestinal, and it can even be asymptomatic. The classic symptoms include chronic diarrhea, steatorrhea, abdominal distension, pain, weight loss and anemia; In children it is also common to have growth retardation. It is currently the most common chronic disease, with a prevalence of 0.7 to 2.0% in the general population and 15 to 20% in first-degree relatives. Celiac disease has a high incidence and, however, there is currently no alternative therapy to gluten-free diet for these patients. In addition, gluten intake and celiac disease are associated with the development of other disorders such as Down syndrome, diabetes mellitus type 1, dermatitis herpetiformis, myopathy, multiple sclerosis, arthritis, autism, schizophrenia, depression, lymphomas or ataxia.
    [0010]
    [0011] Gluten is the environmental factor that triggers celiac disease and associated disorders. It is a set of proteins found in cereal seed such as wheat, barley, rye, spelled, triticale and possibly oats.
    [0012]
    [0013] Wheat gluten proteins are called gliadins and glutenins and have a high content of the amino acid proline, which protects them from proteolytic degradation by part of the human gastrointestinal enzymes. The incomplete digestion of gluten in the intestine, generates two types of peptides involved in the development of celiac disease, are called toxic peptides and immunogenic peptides.
    [0014]
    [0015] The toxic peptides are capable of rapidly inducing damage to the intestinal mucosa by activating the innate immune response of the subject. The most studied are the 19-mer peptide (PGQQQPFPPQQPYPQPQPF), which corresponds to the p31-49 region of a-gliadin and the 13-mer peptide (PGQQQPFPPQQPY), which is a shorter fragment of the previous one and corresponds to the p31 region. -43 from a-gliadin (Sturgess et al., 1994).
    [0016]
    [0017] The immunogenic peptides are those protein fragments capable of activating the adaptive immune response, of which the most studied is the 33-mer (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF), which corresponds to the p57-89 region of the α-gliadin. This peptide is able to induce a strong activation of the T cells of celiac patients and, due to its high proline content (13 of its 33 amino acids), it is very resistant to gastrointestinal digestion (Shan et al., 2002).
    [0018]
    [0019] Under normal conditions the intestinal epithelium is impermeable to these peptides. However, celiac patients have increased intestinal permeability, so that toxic and immunogenic peptides reach the lamina propria of the small intestine and trigger the pathogenesis of celiac disease inducing innate and adaptive immune responses.
    [0020]
    [0021] The innate immune response is caused due to the fact that the toxic peptides of gluten very quickly cause an increase in the expression of interleukin 15 (IL-15), which induces the activation and massive increase of intraepithelial lymphocytes that, when activated, express the NKG2D receptor and transform into Natural Killer (NK) cells. These activated NK cells become cytotoxic causing tissue damage, as they destroy the enterocytes that express molecules of the MHC I type A (MHCA) complex on their surface. The adaptive immune response is slower and begins with the deamination of gluten immunogenic peptides that reach the lamina propria by tissue transglutaminase (TGt). TGT transforms into glutamate the abundant glutamine residues present in the gluten peptides, giving the peptides a negative charge that favors their binding to HLA DQ2 / DQ8 molecules located in the membrane of the antigen-presenting cells. In this way, immunogenic peptides activate TCD4 + lymphocytes restricted by HLA DQ2 / DQ8, which causes the release of proinflammatory cytokines such as interferon gamma, activation of metalloproteinases and other mediators of tissue damage, and the stimulation of B cells that produce anti-gliadin and anti-TGT antibodies.
    [0022]
    [0023] Currently the only completely effective treatment for subjects suffering from celiac disease is to carry out a gluten-free diet for life, which has many disadvantages such as an increase in price, lower palatability and risk of cross contamination between foods.
    [0024]
    [0025] In the agri-food industry, strategies are being developed to reduce the presence of gluten peptides in foods through the genetic manipulation of certain wheat varieties and the use of enzymes and lactic bacteria endowed with proteolytic activity during the fermentation processes of cereals. In this way, it is intended to introduce improvements in the diet of celiac patients and to provide them with a greater variety of products (Rizzello et al., 2007). However, it is very difficult to ensure that a wheat variety, or a fermentation process completely eliminates all the toxic and immunogenic peptides that are generated from gluten.
    [0026]
    [0027] Since genetically modified or manipulated foods during their fermentation neither prevent nor treat the disease, therapeutic alternatives are being developed as compounds inhibitors of the enzyme TGt (WO2007025247), antibodies capable of capturing the peptides derived from the gliadins (US20070184049 A1), compounds which block the binding sites of the gluten peptides to the HLA-DQ2 or HLA-DQ8 molecules (US20070161572 A1) or administration of recombinant regulatory cytokines (Salvati et al., 2005). These strategies involve the modification of molecules involved in multiple biological processes, so that their manipulation can lead to unwanted side effects.
    [0028]
    [0029] Other alternatives that are currently under investigation include the oral administration of proteolytic enzymes obtained from plants or microorganisms to accelerate the digestion of gluten peptides (Gass et al., 2007). However, no in vivo studies have been carried out to demonstrate its efficiency in individuals who ingest gluten as it is present in food. The effects of these enzymes are highly dependent on the time of intake and only reduce the threshold of gluten toxicity.
    [0030] Despite efforts to maintain a gluten-free diet, their traces are often present in the daily life of the patient, for example, whenever they eat food outside their controlled environment, since most people or restaurants can not ensure a complete absence of gluten in their food or culinary processes or have foods certified by FACE (Federations of Celiac Associations of Spain). This means that patients can not go outside their controlled environment to avoid eating foods with traces of gluten that cause an immune response.
    [0031]
    [0032] There is therefore a need to identify substances that can be included in compositions, foods or pharmaceutical compositions to be administered to people suffering from celiac disease or any of its associated disorders, and therefore allow them to eat foods containing traces of gluten, without This negatively affects your health.
    [0033]
    [0034] The use of microorganisms as additives in compositions for administration to celiac patients is a new approach in the treatment and prevention of celiac disease, since it has been seen that there are microorganisms capable of metabolizing gluten.
    [0035]
    [0036] For a microorganism to be used in a composition for the treatment or prevention of celiac disease, it is necessary that the microorganisms meet a series of requirements:
    [0037] (i) metabolize gluten;
    [0038] (ii) avoid the inflammatory activity on the intestinal cells of the peptides derived from gluten;
    [0039] (iii) are stable and resistant to the pH and concentration of bile salts of the gastrointestinal tract;
    [0040] (iv) are safe for the individual;
    [0041] (v) have a high capacity of adhesion to the intestinal epithelium.
    [0042]
    [0043] A composition comprising a microorganism that meets these characteristics would be a breakthrough in the diet of patients, since it could be used as an ingredient in compositions to be administered to these people, since it would allow them to eat foods containing traces of gluten without suffering the effects of the disease and improving their quality of life.
    [0044] WO 2009/080862 A1 describes the use of the microorganism Bifidobacterium longum IATA ES-1 in pharmaceutical and food compositions intended for the treatment or prevention of food allergies, preferably celiac disease. This document is considered the state of the art closest to you. However, it only shows the induction in the production of regulatory cytokines (IL-10 and TGF-P) by the strain in peripheral blood mononuclear cells of healthy individuals (PBMC) and the hydrolysis of peptides of 2-4 amino acids comprising a proline. Therefore, this document lacks sufficient results to guarantee that the strain is capable of using gluten as a nitrogen source, acting on enterocytes of the small intestine or digesting some of the peptides associated with the disease (19-mer and / or 33- mer) and also be safe.
    [0045]
    [0046] It is therefore necessary to find alternative substances that can be added to pharmaceutical compositions, foods or drugs to be administered to people suffering from celiac disease or any of its associated disorders.
    [0047]
    [0048] DESCRIPTION OF THE INVENTION
    [0049]
    [0050] The present invention relates to a microbial strain selected from Lactobacillus rhamnosus L8.6 (CECT 9514), Lactobacillus rhamnosus N4.7 (CECT 9515) and Bifidobacterium breve N24.5 (CECT 9516), or a combination thereof.
    [0051]
    [0052] In one embodiment of the invention, the selected microbial strain of Lactobacillus rhamnosus L8.6, Lactobacillus rhamnosus N4.7 and Bifidobacterium breve N24.5 or a combination thereof, is for use as a medicament.
    [0053]
    [0054] Another subject of the invention relates to a microbial strain selected from Lactobacillus rhamnosus L8.6, Lactobacillus rhamnosus N4.7 and Bifidobacterium breve N24.5 or a combination thereof, for use in the treatment of disorders related to the intake of gluten . In a preferred embodiment, the disorder related to gluten intake is celiac disease.
    [0055]
    [0056] Another embodiment of the invention relates to the use of the microbial strain selected from Lactobacillus rhamnosus L8.6, Lactobacillus rhamnosus N4.7 and Bifidobacterium breve N24.5, or a combination thereof, as an additive in functional foods, probiotic foods, foods symbiotics, food supplements or food nutraceuticals
    [0057]
    [0058] In another embodiment, the invention relates to functional foods, probiotic foods, symbiotic foods, food supplements or nutraceuticals comprising at least one microbial strain selected from Lactobacillus rhamnosus L8.6, Lactobacillus rhamnosus N4.7 and Bifidobacterium breve N24.5, or a combination thereof, for use in the treatment and / or prevention of disorders related to gluten intake. In a preferred embodiment, the disorder related to gluten intake is celiac disease.
    [0059]
    [0060] Another embodiment of the invention relates to a pharmaceutical composition comprising at least one of the microbial strains of the invention. In a preferred embodiment, the composition is used in treatment and / or prevention of disorders related to the intake of gluten. In a more preferred embodiment, the disorder related to gluten intake is celiac disease.
    [0061]
    [0062] Another embodiment of the invention relates to a probiotic composition, functional food, symbiotic food, food supplement or nutraceutical food, comprising at least one of the microbial strains described above. In a preferred embodiment, the composition, food or supplement is used in treatment and / or prevention of disorders related to the intake of gluten. In a more preferred embodiment, the disorder related to gluten intake is celiac disease.
    [0063]
    [0064] The microbial strains of the invention are found in any of the above compositions in a proportion of between 0.1% and 99.9%, preferably between 1% and 90% and more preferably between 10 and 90%. The strains of the invention can be combined with each other or with other microorganisms to improve their protective and metabolic properties through synergistic or complementary actions, such as the increase of the total synthesis of regulatory cytokines and their types, the increase of the inhibitory capacity against bacteria pathogens and the increase of the contribution of enzymes that favor the digestion of proteins and / or gluten peptides increasing their total concentration or increasing their type and specificity.
    [0065]
    [0066] Another embodiment relates to the use of at least one probiotic microbial strain selected from Lactobacillus rhamnosus L8.6, Lactobacillus rhamnosus N4.7 and Bifidobacterium breve N24.5 in the manufacture of medical-pharmaceutical compositions, probiotic compositions, functional foods, symbiotic foods, supplements food or nutraceuticals related to the treatment and / or prevention of disorders related to gluten intake. In a more preferred embodiment, the disorder related to gluten intake is celiac disease.
    [0067]
    [0068] The amount of at least one microbial strain of the invention in pharmaceutical compositions, probiotic compositions, functional foods, synbiotic foods, food supplements is between 105 colony forming units (cfu) and 1013 cfu per gram or milliliter of composition, food or supplement , preferably between 10 6 and 10 12 and more preferably between 10 7 and 10 10 cfu per gram or milliliter.
    [0069]
    [0070] Another object of the present invention relates to a method for the prevention and / or treatment of disorders related to the intake of gluten, preferably celiac disease, which comprises administering a pharmaceutically acceptable amount of a composition comprising at least one microbial strain. probiotic selected from Lactobacillus rhamnosus L8.6, Lactobacillus rhamnosus N4.7 and Bifidobacterium breve N24.5; or administering the probiotic composition, functional food, symbiotic food, dietary supplement or nutraceutical food, comprising at least one probiotic microbial strain selected from Lactobacillus rhamnosus L8.6, Lactobacillus rhamnosus N4.7 and Bifidobacterium breve N24.5; or the pharmaceutical composition comprising said strains.
    [0071]
    [0072] For the purposes of the present invention, the following terms are defined:
    [0073]
    [0074] Probiotic microbial strain: For the purposes of the present invention refers to strains of live bacteria that, when administered to a subject, have a beneficial effect on the health of said subject.
    [0075]
    [0076] Disorders related to gluten intake: The diseases related to gluten intake are, for example and without limitation, celiac disease, non-celiac gluten sensitivity (NG-SG), wheat allergy or irritable bowel syndrome (IBS). and associated effects such as allergy, autism, ataxia, diabetes or multiple sclerosis, among others.
    [0077]
    [0078] Celiac disease (or celiac disease): Celiac disease (CD) is an immune-mediated systemic disease, consisting of an intolerance to gluten proteins that leads to severe atrophy of the mucosa of the upper small intestine. As Consequently, a defect in the utilization of nutrients at the level of the digestive tract is established.
    [0079]
    [0080] Pharmaceutical composition (or medical-pharmaceutical composition): Composition comprising one or more drugs (chemically active substance that exerts its effect on the organism) presented for industrial or clinical use and intended for use in people or animals, endowed with properties that they allow the best pharmacological effect of its components in order to prevent, alleviate or improve the state of health of the sick people, or to modify physiological states.
    [0081]
    [0082] Probiotic composition: for the purposes of the present invention, the term probiotic refers to the use of live microorganisms that are added to compositions or foods (milk, yoghurts, etc.), dietary supplements (in the form of capsules, tablets, pills, powder) , etc.) or others, which remain active and exert their physiological effects on the subject that ingests the food or similar product contained in said probiotic. Ingested in sufficient quantities, they have beneficial effects. The microorganisms of the present invention can be found alive or lyophilized, maintain their biological activity in the intestine and ingested in adequate amounts would exert a beneficial effect on individuals with diseases related to the intake of gluten, reducing their risks and improving their health status.
    [0083]
    [0084] Functional food: Foods that are formulated to provide, beyond their usual nutritional value, a beneficial effect on reducing risks and improving the health status of patients with diseases. In the present case, diseases related to the intake of gluten.
    [0085]
    [0086] Symbiotic food: Functional foods that contain a mixture of prebiotic and probiotic food products. They usually contain a prebiotic component that favors the effect of the probiotic component.
    [0087]
    [0088] Dietary supplement or food: food products whose purpose is to supplement the normal diet and consisting of concentrated sources of nutrients or other substances that have a nutritional or physiological effect. In the case of the use of the microorganisms object of the present invention, a dietary or nutritional supplement would include in its composition at least one of the microorganisms in order to supplement the diet with healthy purposes and, in this specific case, in order to exert beneficial effects on patients with diseases related to gluten intake, reducing their risks and improving their health status.
    [0089]
    [0090] Nutraceutical food: highly concentrated bioactive natural substances that, although they are present in food, are processed to eliminate the excess and leave the beneficial part. Being concentrated and in doses higher than natural food, have a favorable effect on health much greater than that of food as it occurs in nature. In this case, it would exert beneficial effects in patients with diseases related to gluten intake, reducing their risks and improving their health status.
    [0091]
    [0092] Additive: Substance that is added to others to give them qualities that they lack or to improve what they have. In the field of food, are substances that do not constitute a food or have nutritional value, but their addition to food and drink modify their organoleptic characteristics, improve their conservation, texture or enhance some characteristic of the food itself.
    [0093]
    [0094] The concept of probiotic compositions, functional foods, symbiotic foods, dietary or nutritional supplements and nutraceuticals includes, without limitation: dairy products, such as yoghurts, juices, solid foods, as well as tea products, herbalists and parapharmacy, such as as vitamin complexes or nutritional supplements.
    [0095]
    [0096] The strains and compositions comprising them of the present invention are characterized in that they can be used in the treatment and / or prevention of disorders related to the intake of gluten.
    [0097]
    [0098] The strains object of the present invention have the ability to metabolize gluten, which makes them excellent candidates to be included in compositions and food products of celiac people.
    [0099]
    [0100] The treatment of celiac disease or other disorders related to gluten intake prevents the occasional consumption of this protein, causing the harmful effects in the organism indicated above. In this way, the problems suffered by patients with small transgressions of the diet due to the possible mislabeling of gluten-free foods or cross contaminations are avoided. which is a great improvement in the quality of life of people suffering from these diseases.
    [0101]
    [0102] For a microorganism to be considered adequate to metabolize and decrease the concentration of gluten, it must have peptidase activity that hydrolyzes gliadin and other gluten-toxic and immunogenic peptides. The microorganisms of the present invention, or compositions comprising them, can be used in the prevention of disorders related to the intake of gluten, since they are capable of hydrolyzing the toxic and / or immunogenic peptides of the gluten responsible for the activation of the immune response. . In a more preferred embodiment, the microorganisms are capable of hydrolyzing the 19-mer and / or 33-mer peptides. Taking into account the model of the two signals proposed for the development of the pathogenesis of celiac disease (Bernardo, 2008), if these peptides are efficiently hydrolyzed the activation of the innate immune response can be avoided and, as a consequence, it is not triggered the adaptive immune response, thus preventing the pathogenesis of celiac disease from beginning.
    [0103]
    [0104] It is necessary that the strains have anti-inflammatory activity on the intestinal cells. A measure of the anti-inflammatory activity of a compound is the decrease in the release of cytokine IL-8 caused by the presence of a toxic inflammatory compound. The combination of these strains with each other, or with others possessing peptidases of different specificity, allows their action to be complemented, favoring the complete degradation of the toxic and immunogenic epitopes. The three strains described have anti-inflammatory activity.
    [0105]
    [0106] In order for the microorganisms to be suitable for inclusion in compositions for oral administration, it is essential that the microorganisms resist transit through the gastrointestinal tract: they are stable at acidic pH, resistant to gastric and pancreatic juices and at different concentrations of bile salts. In this way, microorganisms can reach the intestine and exert their effect there. The three strains described in the present invention are resistant to the conditions of the gastrointestinal tract.
    [0107]
    [0108] Another requirement of the strains of the present invention is that they are safe, since they are to be administered, directly or comprised in compositions, to human subjects. The safety of a strain can be determined by analyzing virulence factors, such as hyaluronidase activity, elastase activity, activity gelatinic, or hemolytic activity. In addition, the strains should not show resistance to antibiotics.
    [0109]
    [0110] Another characteristic of the strains is the adhesion to the intestinal epithelium for the microorganism to increase the residence time in the intestine and, therefore, the time in which it can exert its effect. The three strains described in the present invention have the ability to adhere to the intestinal epithelium.
    [0111]
    [0112] All these properties guarantee the persistence and prolonged effectiveness in the place in which the strains must carry out their effect, the intestine. They also guarantee its use in the form of probiotic, functional foods, supplements, nutraceuticals and drugs for the reduction of risks and improvement of the state of health and quality of life of the subjects with celiac disease, as well as that of other disorders associated with the ingestion of gluten. Finally, microorganisms have the added value of being able to inhibit the growth of pathogenic microorganisms.
    [0113]
    [0114] The strains of the invention metabolize gluten and have anti-inflammatory activity against gliadin and, therefore, can protect celiac patients on a gluten-free diet from small trace contamination of gluten that can be found in their daily life.
    [0115]
    [0116] Strains Lactobacillus rhamnosus L8.6 (CECT 9514), Lactobacillus rhamnosus N4.7 (CECT 9515) and Bifidobacterium breve N24.5 (CECT 9516) or compositions that comprise them, constitute an alternative for the treatment and prevention of celiac disease and Associated disorders due to their intrinsic characteristics, not described to date in another compound. The advantages of the strains are:
    [0117]
    [0118] - Anti-inflammatory activity
    [0119] - Hydrolysis of gluten toxic and immunogenic peptides
    [0120] - Biosecurity: absence of virulence factors and resistance to antibiotics - Resistance to gastrointestinal stress
    [0121] - Antibacterial activity against pathogenic microorganisms
    [0122] - Adhesion to intestinal cells.
    [0123]
    [0124] Unless defined otherwise, all the technical and scientific terms used herein have the same meaning as those customarily understood by a person skilled in the field of the invention. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. Throughout the description and the claims the word "comprises" and its variants, are not limiting and therefore do not intend to exclude other technical characteristics, additives, components or steps. The term "comprises" also includes the term "consists".
    [0125]
    [0126] BRIEF DESCRIPTION OF THE FIGURES
    [0127]
    [0128] Figure 1. Measurement of the anti-infamatory activity of the isolated microorganisms. The anti-inflammatory activity of the microorganisms L. rhamnosus L8.6, L. rhamnosus N4.7 and B. breve N24.5 is determined according to the average of the amount of cytokine IL-8 released. The white bars correspond to the HT-29 cells (white), the black bars correspond to the HT-29 cells 7 mg / mL PT-gliadin (positive control), the gray bars correspond to the co-culture of HT-29 cells with each microorganism, the grated bars black and gray correspond to the co-culture of HT-29 cells, PT-gliadin and each microorganism, the bars with white and gray squares correspond to the co-culture of HT-29 cells and each microorganism inactivated by heat and the bars with black and gray squares correspond to the co-culture HT-29 cells, PT-gliadin and each microorganism inactivated by heat (* p <0.05).
    [0129]
    [0130] Figure 2. Hydrolysis capacity of the gluten toxic peptides. Chromatograms of the hydrolysis of the 19-mer toxic peptide by the microorganisms L. rhamnosus L8.6 and L. rhamnosus N4.7 and the negative control without incubation with any strain.
    [0131]
    [0132] Figure 3. Hydrolysis capacity of gluten immunogenic peptides. Chromatograms of the 33-mer gluten immunogenic peptide hydrolysis by the microorganisms L. rhamnosus L8.6, L. rhamnosus N4.7 and B. breve N24.5 and the negative control without incubation with any strain. The arrows correspond to the peak corresponding to the 33-mer peptide.
    [0133]
    [0134] Figure 4. Adhesion capacity of microorganisms to intestinal cells. Adhesion capacity of the microorganisms Lactobacillus rhamnosus L8.6, Lactobacillus rhamnosus N4.7 and Bifidobacterium breve N24.5 to the intestinal epithelial cells Caco-2 (black bars). The effect exerted by mucin on the capacity of adhesion to intestinal cells (white bars) is shown. The adhesion is expressed as the percentage of microorganisms adhered with respect to the total number of microorganisms added (* p <0.05).
    [0135] DEPOSIT OF MICROORGANISMS UNDER THE BUDAPEST TREATY
    [0136]
    [0137] The microorganisms used in the present invention have been deposited in the Spanish Type Culture Collection (CECT), located in Building 3 CUE, Parc Científic Universitat de Valencia. Professor Agustín Escardino 9, 46980 Paterna (Valencia, Spain):
    [0138]
    [0139] - CECT 9514: Lactobacillus rhamnosus, L8.6, deposited on November 28, 2017. - CECT 9515. Lactobacillus rhamnosus N4.7, filed on March 27, 2018. - CECT 9516: Bifidobacterium breve N24.5, filed on 27 March 2018
    [0140]
    [0141] EXAMPLES
    [0142]
    [0143] The purpose of the examples indicated below serves to illustrate the invention, without however limiting the scope thereof.
    [0144]
    [0145] Example 1: Isolation and identification of microorganisms that metabolize gluten
    [0146]
    [0147] Samples of breast milk were obtained from healthy voluntary mothers and faecal samples from their healthy children over several periods: 0-5 days from birth and 3 4 months from birth.
    [0148]
    [0149] The samples of breast milk were collected in sterile tubes by manual extraction or by breast pumps, previously cleaning the nipple with soap and water. The tube was completely filled with breast milk to minimize contact with oxygen. The fecal samples were collected directly from the diaper in the shortest time possible after the deposition. They were collected directly by dragging a sterile 125 mL bottle through the diaper and closing it as quickly as possible. Immediately afterwards, the canister with the sample was placed in a 2 liter airtight plastic container together with an envelope of ascorbic acid ( Anaerogen, Oxoid) to generate anoxia in the sample.
    [0150]
    [0151] The samples were processed under aseptic conditions in a biological safety cabinet. The samples of breast milk were diluted in NaCl-cysteine (NaCl 9 g / L with 0.5 g / L cysteine) in a ratio of 1: 5 (v / v). The diluted samples were homogenized with vortex. The fecal samples were diluted in a ratio of 1: 5 (w / v) in NaCl cysteine, were weighed inside the biological safety cabinet and, whenever there was sufficient quantity, the inner part of the sample was used to avoid possible contamination. The diluted fecal sample was homogenized with a Stomacher 80 (Seward) digester.
    [0152]
    [0153] For the isolation of the bacteria from the homogenized samples serial dilutions 1/10 were prepared in NaCl-cysteine. The homogenized breast milk samples were diluted to 10-2 and the homogenized fecal samples were diluted to 10-6. Next, 100 pL of each dilution was plated in duplicate in plates of solid MCG-3 culture medium (Caminero et al., 2014). The MGC-3 medium is a culture medium that carries gluten as the main source of nitrogen and was developed for the isolation and growth of bacteria capable of metabolizing gluten. Each of the replicas was incubated in an airtight container, one with anoxic conditions (without oxygen, generated with Anaerogen, Oxoid envelopes) and the other with microxic conditions (low oxygen concentration, between 2-10%, generated with Campygen envelopes ). , Oxoid). The containers were incubated for 48 hours at 37 ° C.
    [0154]
    [0155] After that time, the MGC-3 plates were analyzed. The morphology of the colonies of the bacteria present in each plate was observed. All the different morphologies that could be differentiated were selected.
    [0156]
    [0157] To determine if a microorganism is capable of metabolizing gluten, it must meet at least one of the following conditions (Caminero et al., 2014):
    [0158]
    [0159] (i) The microorganism does not grow when the gluten is removed from the culture medium in which it has been isolated. This is analyzed with MSG-3 medium, which has the same composition as the MCG-3 medium, but does not carry gluten.
    [0160] (ii) The microorganism grows in a culture medium whose only source of nitrogen is gluten. This was proven by growing the microorganisms in MCG-1 medium. (iii) The microorganism exhibits gluten-plaque activity, that is, it is capable of degrading extracellular gluten.
    [0161]
    [0162] Colonies of bacteria were observed that metabolized gluten according to the above characteristics, were purified and planted four times in isolation by striae exhaustion, to ensure that there was no contamination with other bacteria. Once the microorganisms were isolated, they proceeded to its molecular identification.
    [0163] To identify the isolated microorganisms, their genomic DNA was extracted with the SpeedTools Tissue DNA Extraction Kit (Biotools) and a fragment of 900 base pairs of the gene encoding the 16S rRNA was amplified by PCR. To this end, genomic DNA of each microorganism (between 20 and 50 ng) was used as template, primers oligonucleotides 27F and E939R (final concentration 1 | iM) as described in Baker 2003, with rTaq polymerase (GE Healthcare) . It was checked by agarose gel electrophoresis that the amplified fragment corresponded to the appropriate size (900 bp), it was cloned using the commercial kit Strataclone PCR Cloning (Agilent technologies) in the vector pSC-A-amp / kan. E. coli was transformed with the construct, the plasmid DNA was extracted from the transformants and digested with the Eco RI enzyme to verify that the transformants had incorporated the DNA of interest.
    [0164]
    [0165] To identify the isolated strains, similar sequences were searched in the NCBI GenBank database using the BLAST tool (Altschul et al., 1990). Next, an analysis was made using the program MEGA version 6 ( Molecular Evolutionary Genetics Analysis ) and in this way two isolated strains belonging to the species Lactobacillus rhamnosus (strains L8.6 and N4.7) and a strain belonging to the Bifidobacterium breve species (strain N24.5).
    [0166]
    [0167] Example 2: Anti-inflammatory activity of isolated microorganisms.
    [0168]
    [0169] Assays to determine the anti-inflammatory activity of the microorganisms against gliadin were carried out on cultures of the intestinal epithelial cell line1HT-29 in monolayer between passage 132 and culture 144. The trial consisted of performing a series of co-cultures of the intestinal cells with the microorganisms and PT-gliadin, and measuring the production of inflammatory cytokines in each case.
    [0170]
    [0171] The samples analyzed were the following:
    [0172] - HT-29 cells 7 mg / mL PT-gliadin as a positive control of inflammation.
    [0173] - HT-29 cells 108 cfu / mL of microorganism to measure the effect of the strain on the intestinal epithelium.
    [0174] - HT-29 cells 7 mg / mL PT-gliadin 108 cfu / mL of microorganism to check if the strain reduces inflammation produced by gliadin.
    [0175] - HT-29 cells 108 cfu / mL of microorganism inactivated by heat to measure the effect of the inactivated microorganism.
    [0176] - HT-29 cells 7 mg / mL PT-gliadin 108 cfu / mL of microorganism inactivated by heat, to check if the inactivated microorganism is able to reduce the inflammation produced by gliadin.
    [0177]
    [0178] To simulate the digestion conditions, the gliadin was digested with pepsin and trypsin, giving rise to PT-gliadin, as it occurs in the organism during digestion. To do this, 100 mg of gliadin (Sigma) crushed with a mortar was dissolved in 10 mL water milli-Q pH 2. 34 mg of pepsin (Sigma) was added and the mixture was incubated at 37 ° C for 2 hours with shaking 180 -200 rpm to simulate the conditions of gastric digestion. After incubation, 118.3 mg of Na 2 HPO 4 was added and the pH was increased to 7.9 with 1 M NaOH, as occurs in the transit from the stomach to the intestine. Then 10.3 mg of trypsin (Sigma) was added and incubated again for 2 hours at 37 ° C with 180-200 rpm of shaking to simulate intestinal conditions. After that time, it was incubated for 10 minutes at 99 ° C to inactivate the enzymes and sterilize the mixture. HT-29 cells were cultured at a concentration of 25000 cells / cm2 in 12-well plates treated for cell culture (BD Falcon ™) with 2 mL of McCoy's 5A culture medium supplemented with 10% FBS and 0.4% of antibiotic solution. The culture medium was changed every 2 days for 7 days until the cells were totally differentiated and confluent. After those days, the cell monolayer was composed of 1-2 x 106 HT-29 cells per well. One day before incubation of the co-cultures, the cell monolayer was washed 3 times with PBS pH 7.4 and incubated (for 24 hours at 37 ° C with atmosphere enriched in CO 2 ) with 1 mL of McCoy's 5A medium supplemented with 10% SFB without antibiotics. After incubation, the bacterial strain whose anti-inflammatory capacity was intended to be tested was added to the wells 1-4 x 108 cfu / mL. The strain was added to the well resuspended in McCoy's 5A medium with SFB without antibiotics. The mixture of bacteria and cell monolayer was incubated for 24 hours at 37 ° C with an atmosphere enriched with 5% CO 2 .
    [0179]
    [0180] For cases in which the anti-inflammatory capacity of the inactivated microorganism was tested, it was added to the corresponding well at the same concentration as in the previous case, previously heated at 121 ° C for 60 minutes.
    [0181]
    [0182] PT-gliadin was added to the co-culture at a final concentration of 7 mg / mL. After 24 hours of incubation at 37 ° C in an oven with an atmosphere enriched with 5% CO 2 , the contents of the wells, which contained the cytokines released by the HT-29 intestinal cells, were collected and centrifuged for 15 minutes at 5000 xg to eliminate gliadin from the medium and cell debris. The supernatant was stored at -80 ° C until its processing for the quantification of the released cytokines. For each of the samples and conditions, three independent repetitions were made.
    [0183]
    [0184] For the quantification of the released cytokine IL-8, the BD ™ Cytometric Bead Array ( CBA) Human Inflammatory Cytokines Kit (BD Biosciences) was used following the manufacturer's instructions. The measurement was made using a BD FACS Canto ™ II cytometer , and BD FACS Diva Software was used to interpret the data .
    [0185]
    [0186] The statistical analysis was carried out determining the existence of significant differences (p <0.05) between each test and its respective control by Student's t-test. The tests "microorganism" and "inactivated microorganism" with the "control" were compared, and the tests "gliadin microorganism" and "gliadin inactivated microorganism" with the "gliadin control".
    [0187]
    [0188] Figure 1 shows the anti-inflammatory effect of the microorganisms of the invention, L. rhamnosus L8.6, L. rhamnosus N4.7 and B. breve N24.5 according to the average of the amount of cytokine IL-8 released.
    [0189]
    [0190] Interleukin-8 (IL-8) is a cytokine of the chemokine family, of a proinflammatory nature. Its synthesis is carried out in fibroblasts, endothelial cells, monocytes, macrophages and dendritic cells. It is a potent neutrophil chemotactic factor that regulates the production of adhesion proteins. It is known to amplify the local inflammatory response and stimulate angiogenesis. It is considered that there is anti-inflammatory effect of a strain of the invention when the concentration of IL-8 released by the cell is lower in the presence of the microorganism with respect to the same conditions without microorganism. In this essay it can be seen that the three strains clearly show three effects:
    [0191]
    [0192] i) The three strains have a direct anti-inflammatory effect on intestinal epithelial cells, since they are able to significantly reduce the basal expression of IL-8 when grown directly on HT-29 cells (gray bars versus white bars).
    [0193] ii) The three strains are able to significantly reduce the inflammatory effect caused by PT-gliadin (grated bars versus black bars): Not only do they reduce the inflammatory effect produced by gliadin, but they also reduce the expression of IL-8 by below even the level of IL-8 of the intestinal epithelial cells (white bars).
    [0194] iii) The three strains, even inactivated by heat (bars with white and gray squares), have an anti-inflammatory effect.
    [0195]
    [0196] These results are important because they demonstrate the anti-inflammatory effect of the three strains in the intestinal epithelium in the presence of gliadin peptides, even when the strains have been inactivated. This is important, given that they are probiotic strains whose route of administration is oral that they must pass through the stomach and make their effect in the duodenum. Both environments are physiologically hostile to microorganisms, which could cause the death of a part of the bacteria administered. In this way, it is shown that, even when inactivated, the microorganisms of the invention are capable of exerting an anti-inflammatory effect.
    [0197]
    [0198] Example 3: Hydrolysis capacity of the gluten toxic and immunogenic peptides.
    [0199]
    [0200] Next, it was determined whether the strains isolated in Example 1, in addition to having anti-inflammatory activity (Example 2) were capable of hydrolyzing the toxic and immunogenic peptides generated during the incomplete digestion of the gluten proteins in the intestine.
    [0201]
    [0202] 4.7 pL of 33-mer, or 19-mer toxic peptides at a final concentration of 60 pM were incubated for 24 hours at 37 ° C with 3.4 pL of each strain in PBS pH 7.4, in one volume end of reaction of 40 pL. In turn, 60 pL of PBS and a negative control without microorganism were also incubated as target (4.7 pL of 60 pM peptide 35.3 pL of PBS).
    [0203]
    [0204] After incubation, the reactions were stopped by heating the mixtures at 100 ° C for 10 minutes. They were then filtered through 0.22 pm pore size amicons. They were centrifuged for 2 minutes at 8000 rpm and the eluates were transferred to HPLC vials and kept at 4 ° C until analysis. The samples were analyzed on a Waters Alliance 600 HPLC using a Lichrospher 100 RP18 reverse phase hydrophobic column 25 cm long and 0.4 cm in diameter (Phenomenex). The mobile phases that were used were a mobile phase A with milli-Q water 0.1% trifluoroacetic acid and a mobile phase B with acetonitrile 0.1% trifluoroacetic acid. The injection volume was 10 pL and the injection technique was automatic. The flow was maintained throughout the program at 1 mL / minute and the column temperature was 35 ° C. The method used for the analysis lasts 35 minutes, passing in 20 minutes of 90% of phase A and 10% of phase B to 30% of phase A and 70% of phase B, and subsequently to 100% of phase B that was maintained for 5 minutes. Finally it was passed back to 90% of phase A and 10% of phase B that was maintained for 7 minutes. The absorbance was measured at a wavelength of 215 nm with a Waters 2447 detector. For the analysis of the results, he used the Empower 2 Pro software (Waters version 2005).
    [0205]
    [0206] Figure 2 shows the HPLC chromatograms showing the hydrolysis of the toxic 19-mer gluten peptide by the microorganisms L. rhamnosus L8.6 and L. rhamnosus N4.7 and the negative control.
    [0207]
    [0208] Figure 3 shows the HPLC chromatograms showing the hydrolysis of the 33-mer gluten immunogenic peptide by the microorganisms L. rhamnosus L8.6, L. rhamnosus N4.7, B. breve N24.5 and the negative control .
    [0209]
    [0210] From this experiment it is concluded, firstly, that the strains of the invention L. rhamnosus L8.6 and L. rhamnosus N4.7 are capable of digesting the toxic peptide of gluten 19-mer. This peptide is responsible for the activation of the innate immune response in the body. Taking into account the model of the two signals proposed for the development of the pathogenesis of celiac disease (Bernardo 2008), if these peptides are efficiently hydrolyzed, the activation of the innate immune response can be avoided and, as a consequence, the adaptive immune response, thus preventing the pathogenesis of celiac disease from beginning.
    [0211]
    [0212] In addition, the three strains of the invention are capable of hydrolyzing the 33-mer immunogenic peptide and, therefore, can decrease the concentration of this peptide in the duodenum and prevent the activation of the adaptive immune response.
    [0213]
    [0214] These results demonstrate that the strains L. rhamnosus L8.6, L. rhamnosus N4.7 and B. breve N24.5 hydrolyze the gluten peptides responsible for celiac disease and, therefore, make them excellent candidates for use in pharmaceutical compositions and food products to be administered to celiac patients or related pathologies.
    [0215] Example 4. Biosecurity of microorganisms: evaluation of the presence of virulence factors and resistance to antibiotics.
    [0216]
    [0217] The three microorganisms of the invention belong to species considered QPS ( Qualified Presumption of Safety) by the European Food Safety Agency (EFSA, European Food Safety Authorithy). However, as a precautionary measure, the presence of virulence factors and the antibiotic resistance of the strains isolated in Example 1 were studied.
    [0218]
    [0219] 4.1 Virulence factors
    [0220] The presence of virulence factors in the bacteria was evaluated by testing the presence of enzymatic activities that can cause damage to the host.
    [0221]
    [0222] - Hemolytic activity: The hemolytic activity allows the microorganisms to degrade the hemoglobin present in the red blood cells to dispose of iron, which causes damage to the host.
    [0223] From a pure culture of L. rhamnosus L8.6, L. rhamnosus N4.7 and B. breve N24.5, striae were plated on medium Blood Agar plates. They were incubated for 48 hours at 37 ° C. After the incubation time, if there is hemolytic activity, it is observed directly on the plates due to the formation of a halo of degradation around the colonies. The result of this test is that the three strains do not have hemolytic activity.
    [0224]
    [0225] - Hyaluronidase activity: Hyaluronic acid is a mucopolysaccharide present in human tissues that is depolymerized by the enzyme hyaluronidase. The presence of this activity allows bacteria to invade tissues.
    [0226] From a pure culture of L. rhamnosus L8.6, L. rhamnosus N4.7 and B. breve N24.5, striae were planted on plates of BHIA medium (Brain Heart Infusion Agar) containing 400 pg / mL of hyaluronic acid (Fluka) and 1% bovine serum albumin (BSA) (Sigma). The solution of hyaluronic acid and BSA was added to the BHIA after being sterilized by filtration with 0.22 μm cellulose acetate filters. Plates were incubated 48 hours at 37 ° C. The hyaluronidase activity is detected by adding 2 M acetic acid to the plates and incubating for ten minutes at room temperature. After that time, the plaque becomes opaque except in those areas where there is activity, where translucent zones are observed around the colonies by the degradation of the hyaluronic acid of the medium. The result of this trial showed that none of the three strains has hyaluronidase activity.
    [0227]
    [0228] - Gelatinic activity: This activity is considered a virulence factor because it can cause damage to the extracellular protein matrix of tissues.
    [0229] From a pure culture of L. rhamnosus L8.6 and L. rhamnosus N4.7, striae were plated on MRS medium plates with 3% gelatin (Sigma) and incubated for 48 hours at 37 ° C in microoxic conditions. From a pure B. breve N24.5 culture, striae were plated on MRS medium plates with 2% maltose and 3% gelatin (Sigma) and incubated for 48 hours at 37 ° C under conditions anoxic To detect the gelatinic activity, 30% trichloroacetic acid (TCA) was added to the plate. This acid has the ability to precipitate proteins, so, due to this effect, after a few minutes of adding the plates turn opaque white as a result of the precipitation of gelatin, except around the colonies of the strains that have gelatinic activity, where a transparent halo of degradation is observed. The result of this test showed that none of the three strains object of the invention have gelatinic activity.
    [0230]
    [0231] - Elastic activity: Elastin is a protein that is part of the extracellular matrix of tissues and provides, among other functions, elasticity.
    [0232] From a pure culture of L. rhamnosus L8.6 and L. rhamnosus N4.7, striae were plated on plates of MRS medium with Congo red elastin (Sigma) at 0.1% and incubated for 48 hours at 37 ° C. From a pure B. breve N24.5 culture, striae were plated on plates of MRS medium with 2% maltose and with Congo red elastin (Sigma) at 0.1% and incubated for 48 hours at 37 ° C. After the incubation period, the plates were left at 4 ° C for 3 days. After that time elastic activity was detected by the formation of a halo around the colonies that produce it. The result of this test showed the absence of elastase activity in the three strains of the invention.
    [0233]
    [0234] Table 1 shows a summary of the result of the presence of virulence factors for each strain.
    [0235] Table 1. Result of the biosecurity tests.
    [0236]
    [0237] Activity
    [0238] Microorganism ------------------------------------------------- ----------------------------- Gelatinic elastase Hyaluronidase Hemolytic B. breve N24.5 - - - - L. rhamnosus L8.6 - - - - L. rhamnosus N4.7 - - - -
    [0239]
    [0240] 4.2 Resistance to antibiotics
    [0241] Antibiotic resistance is an undesirable characteristic in genetically transferable bacteria, since it is easily transferable between species. Especially, it is undesirable that microorganisms that are going to be administered to celiac individuals or suffering from associated diseases are resistant to antibiotics, since once in the intestine they can transmit the resistances to other microorganisms. A test was conducted to determine the resistance of strains L. rhamnosus L8.6, L. rhamnosus N4.7 and B. breve N24.5 to antibiotics by determining the minimum inhibitory concentration (MIC) of several antibiotics. The MIC is the lowest concentration of antibiotic from which no bacterial growth is observed.
    [0242]
    [0243] The test was carried out with the antibiotic strips M.I.C.E. (Oxoid), which contain the antibiotic to be tested in a concentration gradient between 0.015 ^ g / mL and 256 ^ g / mL. Seven antibiotics from different pharmacological families were analyzed so that the most important groups of antibiotics were represented. The manufacturer's recommendations were followed and the results obtained were compared with the values of the microbiological cut-off points established by the EFSA for the susceptibility to antibacterial compounds in bacteria used as food additives (EFSA, 2012). Table 2 shows the result for each of the strains. The data obtained is the result of two independent experiments.
    [0244]
    [0245] Table 2. Minimum inhibitory concentration (MIC) of microorganisms for antibiotics.
    [0246]
    [0247] CIM (Hg / mL)
    [0248] Antibiotic ------------------------------------------------- ------------------------------------------ B. Brief N24.5 L. rhamnosus L8.6 L. rhamnosus N4.7 Amoxicillin 1 0.5 1.5 Cefotaxime 12 3 6 Ciprofloxacin 2 0.375 0.375 Erythromycin 1.5 0.023 0.023 Penicillin 0.75 0.375 0.375 Tetracycline 0.18 0.18 0.75 Vancomycin 0.25>256> 256
    [0249]
    [0250] From these experiments it is concluded that strains B. breve N24.5, L. rhamnosus L 8.6 and L. rhamnosus N4.7 do not understand virulence factors nor are they resistant to antibiotics according to the criteria established by the EFSA, which makes safe and suitable to be incorporated into products intended for human and animal consumption.
    [0251]
    [0252] Example 5: Resistance to gastrointestinal stress: resistance to gastric transit, intestinal transit and bile salts.
    [0253]
    [0254] Because the strains of the invention can be part of compositions for oral administration, it is necessary that they are resistant to gastrointestinal stress, that is, they are resistant to all stages of digestion until they exert their anti-inflammatory activity in the small intestine. For this reason, the resistance of each strain to gastric transit, intestinal transit and bile salts was analyzed.
    [0255]
    [0256] 5.1 Resistance to gastric transit
    [0257] From cultures of each strain in stationary phase L. rhamnosus L8.6 and L. rhamnosus N4.7 were incubated in MRS medium at 37 ° C for 48 hours under microoxic conditions and B. breve N24.5 in MRS medium supplemented with 2% maltose at 37 ° C for 48 hours under anoxic conditions. Once the cultures reached their stationary phase, aliquots of 1 mL of each culture were taken and centrifuged at 5000 xg for 5 minutes. The collected cells were washed with PBS at pH 7.4 and each sample was centrifuged at 5000 xg for two minutes. This process was repeated three times. To 0.2 mL of the final suspension of washed bacteria were added 0.3 mL of sterile saline (0.9% NaCl) and 1 mL of a 3 g / L pepsin solution pH 2 or pH 4.5 in saline solution that simulates the gastric juice. In the control experiment, the milliliter of "gastric juice" was replaced by 1 mL of PBS at pH 7.4 Each sample was mixed gently for 5-10 seconds and incubated at 37 ° C. Aliquots of 100 pL were taken. mixtures at times 0, 30, 90 and 180 minutes to determine the total number of viable microorganisms of each strain resistant to gastric juice Serial dilutions were made 1:10 in 0.9% NaCl and plated: L. rhamnosus L8.6 and L. rhamnosus N4.7 in MRS medium and incubated at 37 ° C for 48 hours under microxic conditions, while B. breve N24.5 was seeded in MRS medium supplemented with 2% maltose and incubated at 37 ° C for 48 hours in anoxic conditions. After the incubation time, the colonies of each strain were counted to calculate the cfu / mL and determine its viability.
    [0258]
    [0259] Table 3 shows the result of this test. The resistance of the strains to the gastric juice is expressed as the logarithm of the cfu / mL of the viable microorganisms. The mean of two independent tests is expressed and in parentheses the standard deviation. The asterisks indicate that there are significant differences by the Student's t-test with respect to time 0. ** p <0.01.
    [0260]
    [0261] Table 3. Resistance of microorganisms to gastric transit. NC: No Growth.
    [0262] B. breve L. rhamnosus L. rhamnosus N24.5 L8.6 N4.7
    [0263] 0 min 9.9 (0.16) 9.6 (0.12) 9.1 (0.05) Control 30 min 9.6 (0.03) 9.5 (0.08) 9.1 (0) , 08) (PBS pH 7.4) 90 min 9.6 (0.08) 9.6 (0.06) 9.1 (0.15)
    [0264] 180 min 9.8 (0.02) 9.4 (0.04) 9.4 (0.13) 0 min 10.0 (0.13) 9.5 (0.03) 9.2 (0, 05) 30 min 6.7 (0.07) ** 4.2 (0.09) ** 6.7 (0.04) pH 2 pepsin
    [0265] 90 min NC NC 4.8 (0.05) 180 min NC NC NC
    [0266] 0 min 9.9 (0.08) 9.25 (0.02) 9.3 (0.02) 30 min 9.6 (0.04) 9.3 (0.06) 9.3 (0, 01) pH 4.5 pepsin
    [0267] 90 min 9.4 (0.02) 9.6 (0.05) 9.5 (0.07) 180 min 9.7 (0.04) 9.6 (0.07) 9.4 (0, 14)
    [0268]
    [0269] 5.2 Resistance to intestinal transit
    [0270] To determine the resistance of the microorganisms to the passage through the intestine, a procedure similar to that carried out to study resistance to gastric transit was followed. The difference is that the solution that simulates the gastric juice was replaced by a 3 g / L pancreatin solution in saline at pH 6.5 or pH 8, which simulates the intestinal juice. As a control, the milliliter of solution simulating the "intestinal juice" was replaced by 1 mL of PBS at pH 7. To determine the total number of viable microorganisms, aliquots of 100 pL of each mixture were taken at times 0, 120 and 240 minutes and, in the same way as in the previous case, 1:10 serial dilutions were plated in order to calculate the cfu / mL of each strain and determine its viability.
    [0271] Table 4 shows the result of the test. The resistance of the strains to the intestinal juice is expressed as the logarithm of the cfu / mL of the viable microorganisms. The mean of two independent tests is expressed and in parentheses the standard deviation. The asterisks indicate that there are significant differences through the Student's t-test with respect to time 0. * p <0.05; ** p <0.01.
    [0272]
    [0273] Table 4. Resistance of the microorganisms of the invention to intestinal transit. NC: No Growth.
    [0274] B. breve L. rhamnosus L. rhamnosus N24.5 L8.6 N4.7
    [0275] 0 min 9.9 (0.16) 9.6 (0.12) 9.1 (0.05) Control (PBS)
    [0276] 120 min 9.6 (0.001) 9.06 (0.01) 9.2 (0.02) pH 7.4)
    [0277] 240 min 9.7 (0.02) 9.7 (0.04) 9.5 (0.19) * 0 min 9.9 (0.06) 9.8 (0.06) 9.1 (0) , 15) pH 6.5
    [0278] 120 min 9.7 (0.07) * 9.7 (0.03) ** 9.4 (0.08) pancreatin
    [0279] 240 min 9.2 (0.15) * 9.8 (0.07) 9.4 (0.01)
    [0280] 0 min 10.0 (0.05) 9.4 (0.12) 9.3 (0.03) pH 8
    [0281] 120 min 9.6 (0.03) ** 9.6 (0.12) 9.5 (0.02) pancreatin
    [0282] 240 min 9.8 (0.07) * 9.6 (0.09) 9.4 (0.04)
    [0283]
    [0284] 5.3 Resistance to bile salts
    [0285] From cultures of each strain in stationary phase L. rhamnosus L8.6 and L. rhamnosus N4.7 were incubated in MRS medium at 37 ° C for 48 hours under microoxic conditions and B. breve N24.5 in MRS medium supplemented with 2% maltose at 37 ° C for 48 hours under anoxic conditions. Aliquots of 1 mL of each culture were taken and centrifuged at 5000 xg for 5 minutes. The washed cells were then washed with PBS at pH 7.4 by centrifuging at 5000 xg for two minutes. This process was repeated three times. On the pellet of washed bacteria, 1 mL of the MRS medium was added to L. rhamnosus L8.6 and L. rhamnosus N4.7 and 1 mL of medium MRS supplemented with 2% of maltose to B. breve N24.5 supplemented with different concentrations of bile salts (Sigma) (0.1%, 0.25% and 0.5%). As a control, culture medium without bile salts was added. It was mixed well and incubated at 37 ° C. Aliquots were taken after 1 minute and 4 hours of incubation, serial dilutions were made 1:10 in 0.9% NaCl and sown. Strains L. rhamnosus L8.6 and L. rhamnosus N4.7 were seeded in MRS medium and incubated at 37 ° C for 48 hours under microoxic conditions. B. breve N24.5 was seeded in MRS medium supplemented with 2% maltose and incubated at 37 ° C for 48 hours. hours in anoxic conditions. After the incubation time, the colonies were counted to calculate the cfu / mL and determine its viability.
    [0286]
    [0287] Table 5 shows the result of the test. The resistance of each strain to the bile salts is expressed as the logarithm of the cfu / mL of the viable microorganisms. The mean of two independent tests is expressed and in parentheses the standard deviation. The asterisks indicate that there are significant differences by means of the Student's t test with respect to the control (0% bile salts) at that same time. * p <0.05; ** p <0.01; *** p <0.001.
    [0288]
    [0289] Table 5. Resistance of the microorganisms of the invention to bile salts. NC: No Growth.
    [0290] B. breve L. rhamnosus L. rhamnosus N24.5 L8.6 N4.7 Control (0% 1 min 9.0 (0.04) 10.1 (0.07) 9.6 (0.05) bile salts ) 240 min 9.0 (0.04) 10.5 (0.03) 9.3 (0.09)
    [0291] 0.1% salts 1 min 8.6 (0.10) 9.7 (0.05) ** 9.2 (0.05) biliary 240 min 8.1 (0.04) *** 8.8 (0.07) ** 8.9 (0.03) 0.25% salts 1 min 4.7 (0.08) ** 6.2 (0.08) *** 8.9 (0.007) * bile 240 min 3.7 (0.03) *** 3.8 (0.11) *** 8.3 (0.05) * 0.5% salts 1 min 3.5 (0.05) * * NC 4.2 (0.03) ** biliary 240 min 2.7 (0) ** NC 2.7 (0) **
    [0292]
    [0293] Example 6: Ability to adhere to intestinal cells.
    [0294]
    [0295] The adhesion capacity of the strains of the invention to the intestinal cells is a desirable characteristic for any probiotic microorganism, since it increases the residence time of the probiotic bacteria in the intestine and, therefore, their beneficial effect. In addition, the adhesion of these microorganisms to the intestinal epithelium causes the exclusion of pathogenic microorganisms by competition.
    [0296]
    [0297] The adhesion capacity of the strains of the invention was assessed in the intestinal cell line of human origin Caco-2. This cell line, under standard culture conditions, is able to spontaneously express a morphological and functional differentiation characteristic of mature enterocytes. For this reason, it is an excellent model for studies of adhesion to the human intestinal epithelium.
    [0298] The cells were cultured at a concentration of 25,000 cells / cm 2 in 12-well plates (BD Falcon ™) with 2 mL of EMEM culture medium supplemented with 20% FBS and 0.4% antibiotic solution. The culture medium was changed every 2 days for 10-12 days from sowing, until the cells were totally differentiated and confluent, so that the cell monolayer possessed the optimal conditions to carry out the experiment. The tests were carried out between passes 22 and 30.
    [0299]
    [0300] First, the effect of mucin on the adhesion capacity of microorganisms was evaluated. For this, the previously described protocol was followed, but the Caco-2 cell monolayer was subjected to treatment with porcine type II mucus mucin (Sigma) at the time prior to the addition of the bacterial strain. The presence of mucin better reflects the physiological conditions of the human body, since type II mucins are the main components of the intestinal mucosa. The treatment consisted in adding to the cell monolayer 1 mL of a filtered solution of porcine type II mucin diluted in PBS pH 7.4 at a concentration of 0.5 mg / mL and incubate for 1 hour.
    [0301]
    [0302] Then, the culture medium was removed and the cells were washed 3 times with PBS pH 7.4. After washing, 1 mL of EMEM medium supplemented with 20% FCS without antibiotics was added and incubated for 24 hours at 37 ° C with an atmosphere enriched with 5% CO 2 .
    [0303]
    [0304] After incubation, about 1-4 x 108 cfu of each strain was added to the wells. Bacteria were added resuspended in EMEM medium with SFB without antibiotics and the mixture of intestinal cells and each strain was incubated for 1 hour at 37 ° C with atmosphere enriched with 5% CO 2 . After that time, the cells were washed 3 times with PBS pH 7.4 to remove the non-adherent bacteria. To resuspend the intestinal cells and the attached bacteria the monolayer was disintegrated by adding 200 pL of trypsin-EDTA (0.25% trypsin and 0.53 mM EDTA) and incubated for 10 minutes at 37 ° C. Subsequently, 800 pL of EMEM with SFB without antibiotics was added to inactivate the trypsin and the suspension of intestinal cells and attached bacteria was mixed well.
    [0305]
    [0306] For counting the adhered bacteria, serial dilutions 1:10 with 0.9% NaCl were made, plated on MRS medium plates in the case of L. rhamnosus and medium MRS with 2% maltose for B. brief and calculated the colony forming units per milliliter (cfu / mL). The tests were done in triplicate for each microorganism and adhesion was expressed as the percentage of bacteria adhered to the total of bacteria added.
    [0307]
    [0308] Figure 4 shows adhesion capacity of strains L. rhamnosus L8.6, L. rhamnosus N4.7 and B. breve N24.5 to intestinal epithelial cells Caco-2 in the absence (gray bars) or presence (white bars) of mucin. The adhesion of the microorganisms to intestinal cells is expressed as the percentage of microorganisms adhering to the total of microorganisms added. The data represented are the mean of 3 independent experiments in each case, and the significant differences between adhesion with and without mucin were calculated using the Student's t-test, considering significant differences * p <0.05.
    [0309]
    [0310] As observed in the figure, in the presence of mucin the adhesion of the microorganism to the cells increases significantly, especially in the cases of L. rhamnosus L8.6 and L. rhamnosus N4.7, which makes them very good candidates for used as probiotics with effect in the intestine.
    [0311]
    [0312] Example 7. Antibacterial activity against pathogenic microorganisms.
    [0313]
    [0314] Another mechanism by which probiotic bacteria contribute to host protection is their ability to inhibit the growth of pathogenic microorganisms. Probiotic bacteria can produce antimicrobial compounds that reduce and control the presence of potentially pathogenic microorganisms.
    [0315]
    [0316] To analyze the antibacterial activity of the strains of the invention, two techniques were carried out: the Agar Spot Test technique and the agar diffusion technique of cell-free supernatants (SLC).
    [0317]
    [0318] In the Agar Spot Test technique, 5 pL of the culture (one drop) in stationary phase of each strain was deposited on a plate of solid medium MRS for L. rhamnosus L8.6 and L. rhamnosus N4.7, and on a plate of solid medium MRS supplemented with 2% maltose for B. breve N24.5. Once the drop of each strain was dried, the plate was incubated for 24 hours at 37 ° C. After that time, 7 mL of TSA with 0.5% agar and 0.1% of the pathogenic microorganism Pseudomonas aeruginosa TCD46.1, previously cultured for 24 hours at 37 ° C, was poured on the plates. These plates were incubated for 24 hours at 37 ° C. After that time, growth was observed homogenous of the indicator microorganism throughout the plate. However, around the spot " (growth drop) of each strain a halo of growth inhibition of the indicator microorganism was observed, which indicates that the strains inhibitory activity against pathogenic microorganisms.
    [0319]
    [0320] The agar diffusion technique was carried out in the following way: 1 mL aliquots of cultures of each strain were taken at the end of their exponential phase and centrifuged at 5000 xg for 10 minutes at 4 ° C to obtain the SLC. Plates of solid culture medium TSA were seeded with the indicator pathogenic microorganism P. aeruginosa TCD46.1, previously cultured in liquid medium at 37 ° C for 24 hours. Next, a sterile cotton swab was introduced into the liquid culture and the plate was seeded by dragging the swab throughout the plate in various directions. When the plate was dry, wells of about 7-8 mm in diameter were made in the center and 50 pL of the SLC of the strain to be tested was added to each well. The plates were incubated at 4 ° C for 3 hours for the SLC to diffuse through the agar. After that time, the plate was incubated for 24 hours at 37 ° C. Over time, a homogeneous growth of the indicator pathogenic microorganism was observed throughout the plaque and an inhibition halo around the wells containing the SLC of each of the strains.
    [0321]
    [0322] P. aeruginosa is a bacterium that, in addition to being pathogenic, may be involved in the development of celiac disease (Caminero et al., 2016), hence the great importance of the antibacterial activity of the strains of the invention. It has been shown that strains L. rhamnosus L8.6, L. rhamnosus N4.7 and B. breve N24.5 have antimicrobial activity against P. aeruginosa .
    [0323]
    [0324] In order to know if the inhibitory activity of the strains is due to the production of acids or hydrogen peroxide (H 2 O 2 ) by the microorganisms, the SLCs in which antimicrobial activity was detected were analyzed in the following way:
    [0325] (i) To verify that the antimicrobial activity was due to the formation of acids, a solution of 2 M NaOH was added dropwise to the SLC until its pH was adjusted to 7;
    [0326] (ii) To verify that the activity was due to the production of hydrogen peroxide by the microorganism, each SLC was incubated with catalase (Sigma) at a final concentration of 1 mg / mL for 20 minutes at room temperature.
    [0327]
    [0328] Table 6 shows the antimicrobial activity of the strains L. rhamnosus L8.6, L. rhamnosus N4.7 and B. breve N24.5 against the pathogenic strain P. aeruginosa TCD46.1 analyzed using the techniques "Agar Spot Test" and "Diffusion in SLC agar". The presence of activity is indicated by the sign. The test was done in duplicate to confirm the results. In the cases in which antimicrobial activity was detected in the SLCs, the test was repeated by treating the SLC with NaOH or catalase to check if the antimicrobial activity was due to the production of acids or the production of hydrogen peroxide.
    [0329]
    [0330] Table 6. Antimicrobial activity
    [0331]
    [0332] Pseudomonas aeruginosa TCD46.1 Microorganism -------------------------------------------- ----------------------------____________________ Spot Test SLC SLC NaOH SLC catalase B. breve N24.5 -
    [0333] L. rhamnosus L8.6 -
    [0334] L. rhamnosus N4.7 -
    [0335]
    [0336] From this experiment it is deduced that the three strains are capable of inhibiting the growth of the pathogenic bacterium P. aeruginosa CT46.1 and that the inhibition is due to the production of lactic acid. This corroborates the data obtained in the rest of the examples and demonstrates that the three strains are suitable for use in pharmaceutical or probiotic compositions.
    [0337]
    [0338] BIBLIOGRAPHY
    [0339]
    [0340] Altschul SF1, Gish W, Miller W, Myers EW and Lipman DJ (1990). Basic local alignment search tool. J Mol Biol. 215 (3): 403-410.
    [0341]
    [0342] Baker GC, Smith JJ and Cowan DA (2003). Review and re-analysis of domain-specific 16S primers. J Microbiol Methods. 55: 541-555.
    [0343]
    [0344] Bernardo D (2008). Innate-adaptive interactions in the immune system and its relation to the pathogenesis of celiac disease. Doctoral Thesis University of Valladolid.
    [0345]
    [0346] Caminero A, Herrán AR, Nistal E, Pérez-Andrés J, Vaquero L, Vivas S, Ruiz de Morales JM, Albillos SM and Casqueiro J (2014). Diversity of the cultivable human gut microbiome involved in gluten metabolism: isolation of microorganisms with potential interest for coeliac disease. FEMS Microbiol Ecol. 88: 309-319.
    [0347] Caminero A, Galipeau HJ, McCarville JL, Johnston CW, Bernier SP, Russell AK, Jury J, Herran AR, Casqueiro J, Tye-Din JA, Surette MG, Magarvey NA, Schuppan D, Verdu EF (2016). Duodenal Bacteria From Patients With Celiac Disease and Healthy Subjects Distinctly Affect Gluten Breakdown and Immunogenicity. Gastroenterology 151: 670-683.
    [0348]
    [0349] EFSA (2012). EFSA Panel on additives and products or substances used in animal feed (FEEDAP). Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance. EFSA Journal. 10 (6): 2740.
    [0350]
    [0351] Gass J, Bethune MT, Siegel M, Spencer A and Khosla C (2007). Combination enzyme therapy for gastric digestion of dietary gluten in patients with celiac sprue. Gastroenterology 133: 472-480.
    [0352]
    [0353] Rizzello CG, De Angelis M, Di Cagno R, Camarca A, Silano M, Losito I, De Vincenzi M, De Bari MD, Palmisano F, Maurano F, Gianfrani C and Gobbetti M (2007). Highly efficient gluten degradation by lactobacilli and fungal proteases during food processing: new perspectives for celiac disease. Appl Environ Microbiol. 73 (14): 4499-4507.
    [0354]
    [0355] Salvati MV, Mazzarella G, Gianfrani C, Levings MK, Stefanile R, De Giulio B, Iaquinto G, Giardullo N, Aurichio S, Roncarolo MG and Troncone R (2005). Recombinant human interleukin 10 suppresses gliadin dependent T cell activation in ex vivo cultured intestinal mucosal coeliac. Gut 54 (1): 46-53.
    [0356]
    [0357] Shan L, Molberg O, Parrot I, Hausch F, Filiz F, Gray GM, Sollid LM and Khosla C (2002). Structural basis for gluten intolerance in celiac sprue. Science 297: 2275-2279.
    [0358]
    [0359] Sturgess R, Day P, Ellis HJ, Lundin KE, Gjertsen HA, Kontakou M and Ciclitira PJ (1994). Wheat peptide challenge in coeliac disease. Lancet. 343 (8900): 758-761.
    权利要求:
    Claims (8)
    [1]
    1. Microbial strain selected from Lactobacillus rhamnosus L8.6 (CECT 9514), Lactobacillus rhamnosus N4.7 (CECT 9515) and Bifidobacterium breve N24.5 (CECT 9516) or a combination thereof.
    [2]
    2. Microbial strain according to claim 1, for use as a medicine.
    [3]
    3. Microbial strain according to claim 1, for use in the treatment of disorders related to the intake of gluten.
    [4]
    4. Microbial strain for use according to claim 3, wherein the disorder related to gluten intake is celiac disease.
    [5]
    5. Use of a microbial strain according to claim 1 as an additive in functional foods, probiotic foods, symbiotic foods, food supplements or nutraceuticals.
    [6]
    6. Medical-pharmaceutical composition comprising at least one of the microbial strains according to claim 1.
    [7]
    7. Composition according to claim 6 for use in treatment and / or prevention of disorders related to the intake of gluten.
    [8]
    8. Composition according to claim 7, wherein the disorder related to gluten intake is celiac disease.
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