WO2020040631A1 - System for stabilising human intestinal microbiota - Google Patents

Abstract

The present invention relates to a system comprising compositions and methods for obtaining, stabilising and applying an ex vivo model of gastrointestinal microbiota for use, for example, in the simulation of digestive processes, wherein the inclusion of the stabilised microbiota allows the carrying out of a wide range of studies on populations of microorganisms from donors belonging to a group of interest, according to a characteristic or condition related to age, gender, genotype/phenotype, ethnicity or race, or geographical location, or in relation to a specific change in the state of health, for example, caused by pathogens, nutritional aspects, obesity, renal insufficiency, metabolic disorders, etc. The stabilised microbiota, according to the present invention, can also come from a particular individual, for use in simulations of digestive processes to test therapies related to the formation of metabolites and the impact that they can have on the intestinal microbiota, for example, in cases of inflammatory bowel disease.

Description

 HUMAN INTESTINAL MICROBIOTA STABILIZATION SYSTEM

DESCRIPTION

Technical Field of the Invention

 The present invention corresponds to the technical fields of biotechnology and industrial microbiology, particularly concerns a system composed of compositions and methods for obtaining, stabilizing and applying an ex vivo model of gastrointestinal microbiota, more particularly for its application in in-house models. Colonic fermentation vitro for the simulation of digestion processes and the study of various interactions of the intestinal microbiota.

Background

 The digestive system fulfills the function of transforming the complex substances that are present in food into simpler molecules that can finally be used as a source of energy to carry out the range of metabolic and cellular processes common in the body. However, this primary function is an extremely complex process, in which the different organs that make up the digestive system, such as the mouth (which includes the teeth and the tongue), the pharynx, esophagus, stomach, stomach small intestine, large intestine, rectum and anus, all of them forming the digestive tract also called the gastrointestinal tract or digestive tract; and also involved accessory glands of the digestive tract including the salivary glands, the liver, the gallbladder, the base and the pancreas.

In addition, the digestive system includes vast microbial, heterogeneous populations, composed of archaea, bacteria, fungi, protists and viruses, which are currently considered as a “metabolic organ” that performs per se essential functions in digestion processes, as well as in relation to human health in general. These populations of microorganisms are encompassed in the currently accepted term of "microbiota", and their study has been a subject that becomes more and more relevant as the biochemical and physiological mechanisms that form the basis of the microbiota relationship and the human health In the different anatomical regions of the digestive tract the amount of microorganisms and the species present is variable, for example, the stomach is generally not colonized numerously by microorganisms given the low pH that it has in homeostasis, typically houses up to 1x10 ³ CFU / g, with presence mainly of microorganisms of the lactobacillus, streptococcus, and yeast types, slightly increased in the small intestine the population of microorganisms, settling mostly in the jejunum and ileum in proportions respectively of 1x10 ⁴ and 1 x10 ^6-7 , while in the duodenum the population is rather low due to the presence of bile salts. The highest proportion of the microbiota is established in the large intestine, particularly in the colon, where populations can reach concentrations of up to 1x10 ¹² - 1x10 ¹⁴ CFU / g of colonic content, representing up to 95% of the total microbiota. (Guarner; F., Malagelada, JR., 2003. The bacterial flora of the digestive tract. Gastroenterol Hepatol Vol. 26, Suppl 1: 1-5; Icaza-Chavez, ME, 2013. Microbiota in health and disease. Magazine of Gastroenterology of Mexico Vol.78 (4): 240-248; Williams, CF., Walton, GE., Jiang, L, Plummer, S., Garaiova, /., Gibson, GR. 2015. Comparative analysis of intestinal tract models. Annu Rev Food Sci Technol. Vol. 6: 329-350).

 The relevance of the participation of the intestinal microbiota in the series of biochemical and physiological events that take place during the digestion of food and the subsequent phases of absorption, transport and incorporation into the metabolism of nutrients from them, has generated great interest both in the scientific field and by the industrial sectors related to the production of food, nutraceutical and functional foods, beverages, drugs, medicines and other therapeutic substances. Thus, great effort has been put into designing various strategies to characterize the composition of the intestinal microbiota and its role in digestive processes. It has also been considered essential to have a better understanding of the changes that the intestinal microbiota can undergo in aspects such as diversity, stability and metabolic functionality due to pathological conditions, type of feeding and / or nutritional interventions, medical treatments, among others; since such changes in the intestinal microbiota can have important repercussions on human health. The intestinal microbiota is considered a new factor involved in the etiology of obesity due to its influence on the host's metabolic and immunological functions. (Sanz Y. A., Santacruz, J. D. 2009. Influence of the intestinal microbiota on obesity and metabolism disorders. Acta Pediát Esp. Vol. 67 (9): 437-442)

The population with both childhood and adult obesity has an alteration in the composition of their intestinal microbiota (dysbiosis) that is characterized by a greater amount of Firmicutes and a lower amount of Bacteroidetes, which have been related to the type of diet of the populations {Tremaron V, Bäckhed F. 2012. Functional interactions between the gut microbiota and host metabolism. Natura Sep 13; 489 (7415): 242-9. doi: 10.1038 / nature11552; Estrada-Velasco BI, et al. 2015. Childhood obesity as a result of the interaction between firmicutes and the consumption of Foods with high energy content. Nutr Hosp. 31 (3): 1074-1081 ISSN 0212-1611. doi: 10.3305 / nh.2015.31.3.8302; Sanders ME 2008. Probiotics: definition, sources, selection, and uses. Clin Infect Dis. Vol. 46 (2): S58-S61. doi: 10.1086 / 523341).

 The intestinal microbiota also regulates part of the host's immunity, influences local and systemic responses, so it is considered to have an influence on mild chronic inflammation associated with obesity. Different receptors such as TLRs (Toll-like) are activated in response to microbial stimuli and diet components such as proteins and lipids, triggering signaling pathways that activate different transcription factors such as NFk-b and the synthesis of various cytokines such as TNF- a, IL-6 and IL-1 by inflammation mediators. (Sanz Y. A., Santacruz, J. D. 2009. Influence of the intestinal microbiota on obesity and metabolism disorders. Acta Pediát Esp. Vol. 67 (9): 437-442).

In the state of the art there are a large number of publications in which, although the biological significance of studies conducted directly on the intestinal microbiota can be highlighted, the disadvantages of these types of studies are also weighed. For example, it has been shown that there is a significant inter-individual variation in the intestinal microbiota, so studies in healthy voluntary individuals, or in hospital patients, or in patients with leostomies, or in sudden victims of serious accidents / mortal, would give results with considerable differences; In addition to that it must face a series of ethical considerations. Other factors that may also influence the composition of the intestinal microbiota, include aspects related to age, gender, ethnicity, geography, occupation, anthropometry, eating habits, use of pre / probiotics, nutritional supplements, usual bowel status, use of antibiotics, medications, contact with pets, etc. It has even been observed that the patterns of the intestinal microbiota may also be affected by the diurnal cycles of the microbiota carrier. In addition, there are also the drawbacks that in order to study the intestinal microbiota are the ethical issues involved in how to obtain the intestinal samples to produce populations of microbiota representative of this region of the digestive tract. Attempts to save the issues raised above are disclosed in the prior art, for example, in relation to the implementation of in vivo models (animal models including mice, rats, pigs, etc.); however, due to physiological differences, the results lose biological significance; not counting that they are expensive technologies, and that on the other hand ethical issues are present, for example by the use of animals for experimentation. Another alternative to study the complex bidirectional relationships between the gastrointestinal microbiota and human health consists in the implementation of in vitro models, which involve lower economic costs, lower ethical dilemmas, and in general their operation is relatively simple, allowing relatively rapid obtaining of results. It is also possible to implement these models to simulate in particular some of the digestive tract compartments. The complexity of in vitro models has varied depending on the applications for which they have been designed.

 For example, Batch models are used, such as the simplest designs, in which microorganisms and substrates are incubated in a reactor under controlled conditions. Because the conditions in these types of crops cannot be changed, their use is limited to short-term studies, to prevent microorganisms from reaching a stationary phase due to nutrient depletion and / or accumulation of toxic products that inhibit their growth, and subsequently the viability is lost. (Williams, CF., Walton, GE., Jiang, L, Plummer, S., Garaiova, /., Gibson, GR. 2015. Comparative analysis of intestinal tract models. Annu Rev Food Sci Technol.

Vol. 6: 329-350).

In continuous model designs, it is a question of more precisely mimicking the physicochemical conditions in the gastrointestinal tract; for example, the constant flow of nutrients, the efflux of waste in relation to defined retention times, and the exchange of gases (0 ₂ -C0 ₂ ); and also parameters such as pH and temperature are regulated. Continuous models can be one-step designs (reactor), or consist of several interconnected reactors to simulate different parts of the gastrointestinal system. Examples of this type of in vitro models include the three-step colonic model of Gibson (1988); EnteroMix® by Mákivuokko (2005); and Barner's PolyFermS (2013). (Williams, CF., Walton, GE., Jiang, L, Plummer, S., Garaiova, /., Gibson, GR. 2015. Comparative analysis of intestinal tract models. Annu Rev Food Sci Technol. Voi6: 329-350) .

In dynamic models, a greater similarity is still sought with the physicochemical conditions within the gastrointestinal tract, and include interactions between gastric emptying, stomach pH, secretion, water absorption, removal of digestion products or metabolites of microorganisms, Food transit through the different sections of the digestive tract. Examples of such in vitro models include SHIME (Simulator of the human intestinal microbial ecosystem) by Molly (1993); the TIM (TNO intestinal model) of Minekus (1995-1999); the M-SHIME (Mucosal model -SHIME) by Van den Abbeele (2012); the HMI (Host-microbiota interaction model) of Mazorati (2014), and Kim's Gut-on-a-chip (2012), among others. (Williams, CF., Walton, GE., Jiang, L, Plummer, S., Garaiova, /., Gibson, GR. 2015. Comparative analysis of intestinal tract models. Annu Rev Food Sci Technol. Vol. 6: 329- 350).

 Without prejudice to the foregoing, it is important to note that in order for in vitro models to have a biological significance comparable to that of in vivo models, it is necessary that they have complex, stable microbial populations in their composition, and that they can thus represent Reliable intestinal microbial ecosystem, as well as its metabolic function in the digestive tract and the various processes related to digestion.

Thus, one more alternative is the so-called ex vivo models, in which we want to have microbiota populations with the complexity and diversity of their natural state, and that are isolated from an individual or from a group of individuals with a characteristic of particular interest, for example: in relation to age, gender, genotype / phenotype, ethnicities or races or geographic location; or in relation to some specific alteration of the state of health. In ex vivo models, the isolated microbiota is maintained in artificial environments (in vitro) under controlled conditions. To this end, microbial populations should be partially isolated from their natural habitat, that is, from the gastrointestinal tract, primarily from the colon; However, due to the ethical and technical issues that they would have to face in order to access this part of the human body and take the samples from there, it has been decided to recover the microbiota from the faeces. (Roeselers G., Ponomarenko M., Lukovac S., Wolterboer H., 2013. Ex vivo systems to study host-microbiota interactions in the gastrointestinal tract. Best Practive & Research Clinical Gastroenterology. Vol. 27: 101-113; Wei- Kai W, Chieh-Chang C, Suraphan P, Rou-An C, Ming-Shiang W, Lee-Yan S, Shan-Chwen C. 2018. Optimization of fecal sample Processing for microbiome study - The journey from bathroom to bench. J Formos Med Assoc. Doi: 10. 1016 / ¡.jfma. 2018.02.005; Vogtmann E, Chen J, Kibriya MG, Chen Y, Islam T, Eunes M, AhmedA, NaherJ, Rahman A, AmirA, ShiJ, Abnet CC, Nelson H, Knight R, Chia N, Ahsan H, Sinha R. 2017. Comparison of fecal collection methods for microbiota studies in Bangladesh. Appl Environ Microbiol 83: e00361-17. Doi: 10.1128 / AEM.0061 -17).

The recovery of microbiota from faeces and its subsequent stabilization and maintenance in in vitro systems implies challenges that under various strategies have been tried to save in the procedures disclosed in the prior art. Various methods of preserving fecal samples have proven effective for obtaining nucleic acids with a view to genotypic characterization of the microbiota (Gaci N., Chaudhary PP, Tottey W., Alríc m., Brugére J., 2017. Functional amplification and preservation of human gut microbiota. Microbial Ecology in Health and Disease. Vol. 28, 1308070. doi: 10.1080 / 16512235.2017.1308070)] but with respect to the viability and functionality of the microorganisms recovered from these samples, the picture is different. Although in the maintenance of the microbiota in ex vivo models, careful and controlled management of parameters such as temperature, pH, maintenance of anaerobic environments, retention times, and nutritional substances provided in the culture media used is carried out, etc., to simulate as faithfully as possible the environment-space-time characteristics under which the various members of the intestinal microbiota carry out their metabolic and functional activities, there are critical steps in the preliminary stages of establishing the ex vivo models of Simulation of the gastrointestinal tract, and these critical steps will depend on the level of biological significance that the model will have, that is, that a microbiota with the appropriate population density, sufficiently diverse, stable and metabolically active, is achieved in the simulation model.

 Some of these aspects are addressed, for example, in EP 2750682 B1, which describes an anaerobic micro-ecological system comprising anaerobically cultured human intestinal microbiota, a composition comprising anaerobically cultivated human intestinal microbiota and a method of preparing it . It is mentioned in the document that the composition constitutes a functional culture to restore the normality of a human microbiome affected by diseases; As described herein, the composition is prepared so as to essentially favor the growth of anaerobic bacteria including Bacteroidetes, Firmicutes, Proteobacteria and Actinobacteria, so that their interaction with the damaged intestinal microbiota generates a synergistic effect that promotes restoration of physiological and metabolic functions.

The presence of members of the anaerobic bacterial families mentioned above is also highlighted in WO 2016/139217, which describes the preparation of a human intestinal microbiota composition from faecal samples from healthy donors, for use in therapies of transplantation in the treatment of some condition related to intestinal dysbiosis, derived for example from infections caused by Clostrídium difficile ·, the cultivation of microorganisms is carried out by continuous process in an anaerobic termentator, using as a culture medium a nutritive medium Supplemental with dietary fiber. In US 9,433,651 B2, compositions for microbiota restoration therapy and methods for their manufacture and use are disclosed. The method of manufacturing the composition comprises the collection of a human fecal sample, the addition of a saline solution as a diluent in which a cryoprotectant (Polyethylene glycol) is included, the filtration of the diluted sample for the recovery of a filtrate that is transferred to a sample bag that is sealed at the time of transfer, and subsequently preserved under refrigeration / freezing conditions. The resulting composition, which comprises an effective amount of the fecal microbiota and the cryoprotectant, is used for microbiota restoration therapy by fecal transplantation. And it is mentioned that factors such as storage time, the freeze / thaw technique, the manipulation of the defrosted microbiota, among others are factors that can affect the quality of the sample to be transplanted so that culture techniques are used microbiological to confirm the viability and diversity of the microbiota.

Another example is patent document US 20150037285 A1, which also refers to methods for transferring gastrointestinal microbiota while preserving its viability and bioactivity, even if demanding, anaerobic and non-cultivable organisms are present; Examples of how the manipulation of the gastrointestinal microbiota and the introduction of particular taxa can be used to affect the metabolic state of an individual in which a microbiota transplant is performed, particularly in relation to weight, fat and obesity. It is described in the document that stool samples should be deposited in a container with reduced environment (without oxygen), in a sterile saline solution, water or some other means of aerobic transport; In the case of transport of anaerobic bacteria, the anaerobic environment consists of 90% nitrogen, 5% hydrogen and 5% carbon dioxide in a container that allows these conditions.

And one more case is found in US patent document 2018/0099012 A1, which also deals with the preparation of intestinal microbiota compositions, obtained from faeces of healthy donors, and which are applicable in fecal microbiota transplant therapies in the treatment of intestinal dysbiosis caused for example by infections of pathogenic bacteria such as Clostridium difficile. The method includes the steps of: collecting at least one stool microbiota sample from the donor subject, and within a period of less than 5 minutes after collecting the sample place it in an oxygen-deprived device; but the sample obtained must be mixed with at least one aqueous saline solution containing at least one cryoprotectant and subsequently, the mixture obtained is filtered through a filter comprising pores having a diameter of less than or equal to 0.7 mm and finally store the mixture obtained at a temperature between -15 ° C and -100 ° C.

 However, the need to improve the methods of obtaining, stabilizing and maintaining, at optimal times, of microbial populations that could be considered biologically significant representations of the intestinal human microbiota has persisted. The microbiota obtained for transplantation, according to the methods previously disclosed in the state of the art, was not maintained in culture, but only the optimal conditions were established to preserve it until the moment of its use; with the constant risk that microorganisms were lost due to failures in the microbiological techniques applied during the manipulation of said microbiota.

Brief Description of the Invention

 The invention that will be described hereinafter provides an attractive and cost-effective system, in which different elements that allow the stabilization of the human intestinal microbiota, obtained from stool samples, are integrated, so the involvement of invasive techniques

 The system of the invention is composed of a stool sampling kit designed so that when it is provided to a donor or group of donors, it is easy to collect the samples from which the intestinal microbiota will be obtained. that ensures the viability of the microorganisms that are contained in it.

 Another primary element of the invention consists of a method whose implementation according to the steps and steps that will be described later, allows to effectively achieve in vitro stabilization of human intestinal microbiota from stool samples from a donor or group of donors with at least one characteristic of specific interest for its study, which may correspond for example to an alteration such as obesity, where the IMG is equal to or greater than 30, without comorbidities; or to other types of disorders such as diabetes; inflammatory bowel disease; among other. Even the donor or group of donors can be individuals in an unaltered state including healthy children, healthy adults, healthy athletes, among others.

The method of the invention involves the use of containers where the fermentation reactions inherent in the stabilization of the human intestinal microbiota will be carried out, which will be defined as reactors, which act according to the method of the invention simulating respectively one or more anatomical regions of the digestive tract. As part of the method of the invention, it is necessary to establish in the reactors the conditions of at least pH, stirring, and temperature, in function of the anatomical region of the digestive tract that will be simulated. These values are widely disseminated in the literature, for example it is well known that the pH in the stomach of a healthy adult ranges from 2 to 2.5.

 And another primary element of the invention consists of a culture medium for the stabilization of the microbiota, designed such that without incurring high costs it allows to achieve the stabilization of the intestinal microbiota obtained from stool samples, and also allows to maintain the intestinal microbiota human in vitro conditions even for prolonged periods of time necessary for the study of microbial populations, their interactions, and / or simulations of a range of digestive processes.

 Brief description of the figures

 Figure 1 schematically exemplifies the main components of the stool sampling kit: the containers (1) with a tight-fitting lid (2), where (1 a) corresponds to the sample container under anaerobiosis conditions and (1 b) corresponds to the container for the aerobic sample; in the case of a sample in anaerobiosis, a candle of such size as to have no contact with the final content (3) is incorporated into the inner face of the sealing cap (2); both containers include a volume of culture medium for sampling (4); the device for sample collection (5); and the instructions for sampling (6).

Figure 2 shows a graph corresponding to the microbiota monitoring of patients in a pre-dialysis situation, stabilized according to the method of the invention for a simulation process of ascending colon, transverse colon, and descending colon. The presence of bacteria of the genera Lactobacillus, Bifidobacterium, Salmonella, and Clostridiunr was observed, whose proportions were maintained during the stabilization of the microbiota, with respect to the proportions found in the native microbiota of donor patients

 Figure 3 shows a graph corresponding to the microbiota monitoring of patients undergoing hemodialysis, stabilized according to the method of the invention for a simulation process of ascending colon, transverse colon, and descending colon. The presence of bacteria of the genera Lactobacillus, Bifidobacterium, Salmonella, and Clostridium was observed; whose proportions were maintained during the stabilization of the microbiota, with respect to the proportions found in the native microbiota of the donor patients

Figure 4 shows a graph corresponding to the microbiota monitoring of patients with inflammatory bowel disease, stabilized according to the method of invention for a simulation process of ascending colon, transverse colon, and descending colon. The presence of bacteria of the genera Lactobaciilus, Bifidobacterium, Salmonella, and Clostridium was observed; whose proportions were maintained during the stabilization of the microbiota, with respect to the proportions found in the patients who donated the initial microbiota which is taken as a reference to ensure conditions of microorganisms similar to the patient's intestine.

 Detailed description of the invention

 The invention referred to herein refers to a human intestinal microbiota stabilization system, in which a stool sampling kit for the recovery of the Intestinal microbiota is integrated; a culture medium for stabilizing the intestinal microbiota obtained from stool samples; and, a method for in vitro stabilization of the intestinal microbiota obtained from stool samples; elements that as a whole allow the isolation, stabilization, maintenance and application of the intestinal microbiota in ex vivo models for example for the simulation of digestion processes, where the incorporation of the stabilized microbiota allows a range of studies on populations of microorganisms from donors belonging to a group of interest for any characteristic or condition related to age, gender, genotype / phenotype, ethnicity or race or geographic location; or in relation to some specific alteration of the state of health, for example caused by pathogens, by nutritional aspects, by obesity, renal insufficiency, metabolic disorders, etc. The stabilized microbiota, in accordance with the present invention, can also come from a particular individual for use in simulations of digestive processes, to test therapies that are related to the formation of metabolites and the impact they may have on the intestinal microbiota, for example in cases of inflammatory bowel disease.

 A first aspect of the invention comprises the stool sampling kit (Figure 1), which is designed to ensure the diversity, quantity of the population, and metabolic activity of the aerobic and anaerobic groups. The kit includes for each donation / donor of stool sample:

1) At least two containers, preferably with a capacity of 5 to 50 mL, made of material that can be sterilized, and both with sealed lids (2). In at least one of the containers (1 a) a candle (3) is provided adhered to the inner face of the sealing lid, so that when lighting the candle after depositing the sample and covering the container tightly, it is carried carried out an oxygen to CO2 conversion reaction to generate an anaerobic environment (sample anaerobic); while in at least one container (1 b) there will be an aerobic environment (aerobic sample).

2) A sampling culture medium (4), which includes carbon sources such as yeast extract (1-3 g / L) and peptone (1-3 g / L), inorganic salts such as NaHCO ₃ (0.1- 0.1 g / L), NaCI (0.01-0.1g / L), K ₂ HPO ₄ (0.01 -0.1 g / L), KH ₂ PO ₄ (0.01-0.1 g / L), CaCI ₂ (0.001 -

0.01 g / L), MgSO ₄ 7H ₂ O (0.001-0.01 g / L), nitrogen sources such as L-cysteine (0.1-1g / L), and an iron source that can optionally be selected from ferrous sulfate, ferric chloride, hemin and erythrocyte extract (0.1-0.5 g / L). The sample culture medium (4) is provided in a volume of between 1 and 30 mL, sterile, included in the respective containers with sealed lids (1 a, 1b), so that it acts as a means of transport of stool samples, preserving the viability and metabolic activity of the diversity of microorganisms present in the samples until the time of processing, in a period of up to 8 hours after stool deposition.

Optionally, the kit can also include:

 3) At least one device for sample collection (5), which must be made of material that can be aseptically, preferably in the form of a spoon for the collection of stool samples of different consistency, with a capacity of between 1 and 10 g of sample.

4) A sampling instruction (6), to provide specific indications on how the stool sample should be collected.

A second aspect of the invention comprises the microbiota stabilization culture medium, which for its proper operation according to the invention is provided in two compositions: the first composition corresponds to a base culture medium comprising carbon sources such as yeast extract (1-3 g / L) and peptone (1-3 g / L), inorganic salts such as NaHCO ₃ (0.1-0.1 g / L), NaCI (0.01 -0.1 g / L), K ₂ HPO ₄ (0.01 -0.1 g / L), KH ₂ PO ₄ (0.01-0.1 g / L), CaCI ₂ (0.001-0.01 g / L), MgSO ₄ 7H ₂ O (0.001-0.01 g / L), nitrogen sources as L-cysteine (0.1-1 g / L); and the second composition corresponds to a multivitamin that includes at least per liter: biotin (5000 Ul), pantothenate (10 mg), nicotinamide (5 mg), vitamin B12 (cyanocobalamin) (5 mg), thiamine (100 mg), Benzoic or paraminobenzoic acid (vitamin C) (150 mg), menodione (100 mg). The microbiota stabilization culture medium provides adequate nutrients to preserve the desired characteristics of the microbiota obtained from stool samples for long periods of ex vivo culture, namely diversity, population density and metabolic activity. A third aspect of the invention comprises the method for the in vitro stabilization of the isolated microbiota of stool samples, for later use in one or more vessels that meet the characteristics of a reactor, preferably a flat base with working capacity. ranging from 100 to 1000 mL, in which conditions are established very close to those prevailing in the natural habitat of the microbiota, to maintain the crops even for prolonged periods of time, necessary for conducting various tests, for example in digestion simulation tests, in colonic processes, of the interaction of microorganisms with pathogens, by nutritional aspects, by obesity, renal insufficiency, metabolic disorders, etc. The simulation can be performed by a particular individual to test therapies that are related to the formation of metabolites and the impact they may have on the intestinal microbiota, for example in cases of inflammatory bowel disease.

 The stool microbiota stabilization method comprises the following steps and steps:

 Collection of stool samples.

a) determine on the basis of a characteristic of specific interest of study the donor or group of donors of the faeces; For example, individuals with a defined metabolic characteristic are selected that may correspond to an alteration such as obesity, diabetes, inflammatory bowel disease, etc., or to individuals in an unaltered state such as healthy children, healthy adults, healthy athletes, among others.

b) provide a sampling kit, to the selected individual or donor individuals, that is equipped with at least two containers with a tight-fitting lid (2); each of the containers preferably containing a volume of between 1 and 30 mL of sterile sampling medium; where at least one of the containers can generate an anaerobic environment (container 1 a) by providing a candle (3) attached to the inner face of the tight-fitting lid, so that the donor lights it after deposit the sample, and that when the container (1 a) is sealed tightly, an oxygen to CO ₂ conversion reaction is carried out that propitiates the anaerobic condition suitable for having an anaerobic sample; and where at least one container (1 b) has an aerobic environment that allows an aerobic sample to be obtained; and request that the donor individual or individuals deliver the samples within a period not exceeding 6 hours after stool deposition. C) obtain for each donor / donation at least one aerobic sample, in which the permanence of aerobic microorganisms has been ensured, and an anaerobic sample, in which the permanence of anaerobic microorganisms has been ensured.

II. Processing of stool samples.

a) combining the aerobic (container 1b) and anaerobic (container 1a) samples in a collecting tube, suitable for centrifugal separation of a supernatant phase, in which the microbiota bacteria are contained, of an organic phase, consisting of the tablet formed after centrifugation.

b) verify the viability and diversity of the bacteria present in the microbiota isolated from faeces, by means of at least one of the microbiological techniques already known in the state of the art; for example the use of selective means, the determination of microorganisms by molecular methods, or any other methodology that allows to verify that the isolated microbiota is viable and diverse.

 For the diversity aspect, the presence of at least 4 to 10 bacterial groups of interest will be considered necessary, for example Lactobacillus spp, Bifidobacterium spp,

Clostridium spp, Salmonella spp, Enterobacteriae spp, Prevotella spp, or those that are of particular interest according to the process being carried out; while in the viability aspect it will be considered necessary that the bacterial groups of interest obtained are metabolically active, that is to say that they are capable of reproducing and forming at least one metabolite of interest, for example short chain fatty acids (lactic acid, butyric acid , propionic acid) among others.

 III. Stabilization and maintenance ex vivo of microbiota cultures.

a) prepare one or more vessels that will act as reactors to simulate a specific anatomical region of the digestive tract, preferably with a flat base with a working capacity ranging from 100 to 1000 mL where the microbiota will be stabilized; where the preparation refers to placing a base medium for the stabilization of the intestinal microbiota obtained from stool samples, which can be 10% of the reactor's workload comprising: yeast extract (1-3 g / L ), peptone (1 -3 g / L), NaHCO ₃ (0.1 -0.1 g / L), NaCI (0.01-0.1g / L), K ₂ HPO ₄ (0.01-0.1 g / L), KH ₂ PO ₄ (0.01-0.1 g / L), CaCI ₂ (0.001-0.01 g / L), MgSO ₄ · 7H ₂ O (0.001-0.01 g / L), and L-cysteine (0.1 -1 g / L); and maintain said base medium in sterile conditions;

b) inoculate a volume of supernatant obtained in step ll-a, corresponding to 10% of the total volume of culture medium of step lll-a; where the supernatant is obtained by combining the aerobic (container 1 b) and anaerobic (container 1 a) samples in a tube suitable for centrifugal separation of a supernatant phase, in which they are Contained the bacteria of the microbiota, of an organic phase, consisting of the tablet formed after centrifugation.

c) establish the conditions of at least pH, agitation, temperature, depending on the anatomical region of the digestive tract that will be simulated in the reactor or reactors;

d) keep the culture in incubation at 37 ° C, with constant agitation to keep in contact and interaction all the elements contained in the reactor;

e) every 24 hours verify the pH and add a multivitamin solution that includes at least per liter: biotin (5000 Ul), pantothenate (10 mg), nicotinamide (5 mg), vitamin B12 (cyanocobalamin) (5 mg), thiamine (100 mg), benzoic or paraminobenzoic acid (vitamin C) (150 mg), menodione (100 mg), repeating this operation until the pH stabilizes and is constant for at least 3 consecutive days and the proportion of microorganisms is maintained regarding the proportion of microorganisms in the initial sample; Y

f) carry out a second step of verifying the viability and diversity of the microbiota in a manner similar to that performed in step ll-b.

 Rare to consider that the microbiota has stabilized successfully, at least three criteria must be considered: 1) that it is variable, that is, that it can identify between 4 and 10 bacterial groups of interest in the stabilized microbiota; 2) that it is stable, which is determined when the pH of the different simulated sections remains constant; 3) that microorganisms are not only viable but metabolically active, that is to say that they are able to reproduce and form at least one metabolite of interest. Each part of the stabilization process can be verified using different techniques, different methods and different instruments, according to the needs and conditions of the user.

Finally, if the stabilized microbiota is desired to be preserved, any preservation method described above can be used, for example mixing the stabilized microbiota with 70% glycerol in a 1: 1 ratio, quickly freezing in a rapid freezing system and then being maintained. in freezing at -80 ° C.

Examples of application of the invention

In order to provide elements that allow a greater understanding of the inventive concept that gives rise to the present, examples of application of the invention are described below, with the understanding that they are merely illustrative examples without being intended in any way to limit the scope of the invention. Example 1. Stabilization of intestinal microbiota of patients on pre-dialysis and hemodialysis.

 The intestinal microbiota analysis of each population studied was performed. Five patients in a pre-dialysis situation and 5 patients in a hemodialysis situation were analyzed. In the case of pre-dialysis patients, all patients were positive for the presence of Clostridium perfringes and negative for the presence of yeasts, as was the microbiota of patients on hemodialysis, however the proportion of bacterial groups was shown. with substantial differences as can be seen. In pre-dialysis individuals, the proportion Lactobacillus 26.6%, Bifidobacterium 20%, Salmonella 26%, Clostridium 26.6%; which is taken as a reference to consider the stabilized microbiota, which, as can be seen, keeps the same proportion of the bacteria measured. In the case of hemodialysis patients, the proportion Lactobacillus 23.8%, Bifidobacterium 26.1%, Salmonella 23%, Clostridium 27%, which is maintained in the stabilized microbiota, is saved, as can be verified in Tables 1 and 2 and Figures 2 and 3.

 Table 2 shows the intestinal microbiota analysis of hemodialysis patients, in addition to the 4 groups, Clostridium perfringens and yeasts were evaluated. It was observed that 100% of patients both in pre-dialysis and hemodialysis showed the presence of Clostridium perfringens. None of the patients showed the presence of yeast in stool.

 Table 1. Microbiota analysis in pre-dialysis patients

Table. 2 Analysis of intestinal microbiota of patients undergoing hemodialysis.

Ejemplo 2. Estabilización de microbiota de individuos con enfermedad inflamatoria intestinal

Example 2. Stabilization of microbiota of individuals with inflammatory bowel disease

 Samples were taken from 15 patients who presented inflammatory bowel disease, specifically Nonspecific Inflammatory Ulcerative Colitis (UC) of which the individual analysis was performed (table 3) that was taken as a reference to stabilize the microbiota.

 It can be seen that the proportion of the groups analyzed is maintained in the stabilized microbiota as shown in Figure 4. In the case of patients with UC, there was presence of Clostridium perfringes and yeasts in different patients, so we assume that the microbiota Stabilized will contain both yeast and potentially inflammatory precursor agents such as C. perfringes.

As can be seen, the proportions of the bacterial groups counted are maintained in the stabilized microbiota with respect to the original microbiota, which are maintained in the following proportions: Lactobacillus 21.75%, Bifidobacterium 26.3%, Saimonella 24%, Clostridium 27.3%.

Claims

 1. A stool sampling kit for isolation and stabilization of human intestinal microbiota, characterized in that it comprises:  i) at least two containers (1) with sealing lids (2), a candle (3) being provided adhered to the inner face of the sealing lid in at least one of the containers (1 a); while in at least one container (1 b) there will be an aerobic environment; Y ii) a sampling culture medium (4), which includes carbon sources such as yeast extract (1-3 g / L) and peptone (1-3 g / L), inorganic salts such as NaHC0 ₃ (0.1- 0.1 g / L), NaCI (0.01 -0.1g / L), K ₂ HPO ₄ (0.01 -0.1 g / L), KH ₂ PO ₄ (0.01-0.1 g / L), CaCI ₂ (0.001-0.01 g / L), MgSO ₄ · 7H ₂ O (0.001-0.01 g / L), nitrogen sources such as L-cysteine (0.1-1 g / L), and an iron source that can optionally be selected from ferrous sulfate, chloride ferric, hemin and erythrocyte extract (0.1-0.5 g / L). 2. The kit according to claim 1, characterized in that the containers (1) with a tight-fitting lid (2) preferably have a capacity of 5 to 50 mL, and are manufactured with material that can be sterilized. 3. The kit according to any one of claims 1-2, characterized in that the container (1 a) in which the candle (3) provided on the inner face of the sealing lid is provided is intended to preserve the microorganisms Anaerobic, once the candle is lit after the sample has been deposited and the container is covered tightly, an oxygen conversion reaction to C0 _{2 is carried out} to generate an anaerobic environment.  4. The kit according to any one of claims 1 -2, characterized in that at least one container (1 b) without attached candle will be intended to preserve aerobic microorganisms by keeping the sample in an aerobic environment.  5. The kit according to claim 1, characterized in that the sample culture medium (4) is provided in a volume of between 1 and 30 mL, sterile, in each of the respective containers with hermetically sealed lids. (1 a, 1 b). 6. The kit according to claim 5, wherein the sampling culture medium (4) acts as a means of transporting stool samples to preserve the viability and metabolic activity of the diversity of microorganisms present in the Samples up to 8 hours after stool deposition. 7. The kit according to claim 1, characterized in that the sample collection kit optionally includes at least one device for sample collection (5) made of material capable of being aseptized, and preferably having a teaspoon shape for the collection of stool samples of different consistency.  8. The kit according to claim 1, characterized in that the kit optionally includes a sample collection instruction, in which specific indications are given to the donor or donors on the manner in which the sample collection of stool 9. A method for in vitro stabilization of human intestinal microbiota from faeces comprising the following steps and steps:  I. Collect stool samples by the following steps:  a) determine on the basis of a characteristic of specific interest of study the donor or group of donors of the faeces;  b) providing a sampling kit as described in claim 1, to the selected donor individual or individuals, requesting to deliver the samples in a period not exceeding 8 h after stool deposition;  c) obtain for each donor / donation at least one aerobic sample, in which the permanence of aerobic microorganisms has been ensured, and an anaerobic sample, in which the permanence of anaerobic microorganisms has been ensured;  II. Process the stool samples by the following steps: a) combine the aerobic and anaerobic samples in a collecting tube, suitable for centrifugal separation of a supernatant phase, in which the microbiota bacteria are contained, of an organic phase , which consists of the tablet formed after centrifugation;  b) verify the viability and diversity of the microbiota recovered in the supernatant phase of step (a), by means of at least one of the microbiological techniques already known in the state of the art;  III. Stabilize the isolated microbiota and keep it in ex vivo models through the following steps: a) prepare one or more vessels that will act as reactors to simulate a specific anatomical region of the digestive tract, preferably of base flat with working capacity ranging from 100 to 1000 mL where the microbiota stabilization will be performed; wherein the preparation refers to placing a base medium for the stabilization of the intestinal microbiota obtained from stool samples, which can be 10% of the reactor's workload comprising: yeast extract (1-3 g / L ), peptone (1-3 g / L), NaHCO ₃ (0.1 -0.1 g / L), NaCI (0.01 -0.1 g / L), K ₂ HPO ₄ (0.01-0.1 g / L), KH ₂ PO ₄ (0.01-0.1 g / L), CaCI ₂ (0.001-0.01 g / L), MgSO ₄ · 7H ₂ O (0.001-0.01 g / L), and L-cysteine (0.1-1 g / L); and maintain said base medium in sterile conditions; b) inoculate a volume of supernatant obtained in step ll-a, corresponding to 10% of the total volume of culture medium of step lll-a;  c) establish the conditions of at least pH, agitation, temperature, depending on the anatomical region of the digestive tract that will be simulated in the reactor;  d) keep the culture in incubation at 37 ° C, with constant agitation to keep in contact and interaction all the elements contained in the reactor;  e) every 24 hours verify the pH and add a multivitamin solution that includes at least per liter: biotin (5000 Ul), pantothenate (10 mg), nicotinamide (5 mg), vitamin B12 (cyanocobalamin) (5 mg), thiamine (100 mg), benzoic or paraminobenzoic acid (vitamin C) (150 mg), menodione (100 mg), repeating this operation until the pH stabilizes and is constant for at least 3 consecutive days and the proportion of microorganisms is maintained regarding the proportion of microorganisms in the initial sample; f) carry out a second step of verifying the viability and diversity of the microbiota in a manner similar to that performed in step ll-b.  10. The method according to claim 9, wherein in step 1, the characteristic of specific study interest in the donor or group of stool donors is selected from individuals with a defined metabolic characteristic including obesity, diabetes, disease inflammatory bowel; or to individuals in an unaltered state including healthy children, healthy adults, healthy athletes.  11. The method according to claim 9, wherein in step l-c, the permanence of aerobic and anaerobic microorganisms is ensured by the use of the correct method 12. The method according to claim 9, wherein the microbiological techniques for the verification of step ll-b include the use of means selective; and / or the determination of microorganisms by molecular methods; and / or any other methodology that allows to verify that the isolated microbiota is viable and diverse.  13. The method according to claim 12, wherein the diversity of the microbiota is acceptable if at least 4 to 10 bacterial groups of interest are detected by microbiological techniques.  14. The method according to claim 12, wherein the viability of the microbiota is acceptable if it is detected by microbiological techniques that the bacterial groups of interest obtained are metabolically active, that is to say that they are capable of reproducing and forming at least one metabolite of interest selected. 15. A culture medium for the stabilization of intestinal microbiota obtained from stool samples, characterized in that for its proper functioning it is provided in two compositions, of which the first corresponds to a base culture medium comprising: yeast extract ( 1-3 g / L), peptone (1-3 g / L), NaHCO ₃ (0.1-0.1 g / L), NaCI (0.01-0.1g / L), K ₂ HPO ₄ (0.01-0.1 g / L ), KH ₂ PO ₄ (0.01-0.1 g / L), CaCI ₂ (0.001-0.01 g / L), MgS0 ₄ · 7H ₂ O (0.001-0.01 g / L), and L-cysteine (0.1-1g / L); and the second composition corresponds to a multivitamin that includes at least per liter: biotin (5000 Ul), pantothenate (10 mg), nicotinamide (5 mg), vitamin B12 (cyanocobalamin) (5 mg), thiamine (100 mg), Benzoic or paraminobenzoic acid (vitamin C) (150 mg), menodione (100 mg).