RU2823233C2 - Use of microbial communities for treating humans and animals - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine, namely to compositions for preventing or treating symptoms associated with a gastrointestinal disorder. Disclosed is a composition for preventing or treating symptoms associated with a gastrointestinal disorder, comprising bacterial species that are purified, and wherein the bacterial species include at least one short-chain fatty acid (SCFA) producing bacterial species, and at least one SCFA-metabolising bacterial species, where when the composition is cultured in an acidic medium at pH 6–8 and conditions with low oxygen content of up to 8 g/l, at least one SCFA-producing bacterial species is present in an amount, sufficient to provide an amount of SCFA sufficient to maintain the growth of at least one SCFA-metabolizing bacteria. Composition for preventing or treating symptoms associated with a gastrointestinal disorder, containing types of bacteria, which are purified and are in suspension, is characterized by the above composition. Composition for preventing or treating symptoms associated with a gastrointestinal disorder differs from the above in that it is in powder form.

EFFECT: said group of inventions is suitable for preventing or treating symptoms associated with a gastrointestinal disorder.

26 cl, 12 dwg, 2 tbl, 3 ex

Description

Field to which the invention relates

The present invention relates to a mixture of bacteria belonging to at least 6 or 7 different and specific bacterial species, preferably for use in the prevention or treatment of gastrointestinal disorders. More preferably, said mixture of bacteria is cultured in a fermenter before administering said mixture to an individual for the prevention or treatment of said disorder.

Prior Art

The human and animal gut ecosystem consists of many different habitats and metabolic niches that are colonized by so-called microbiotas containing more than 10 ¹¹ microorganisms per gram wet weight of the contents, predominantly anaerobes (Macfarlane & Macfarlane, 1997). It is now well established that the human or animal gut microbiome plays an important role in maintaining human health and well-being through the release of energy, modulation of the immune system and development of resistance to colonization by opportunistic pathogens (Fuller & Gibson, 1997; Cummings & Macfarlane, 1997) . There is evidence that the interaction of bacteria and their metabolites with the mucosal layer and/or the intestinal wall is an important factor (Barnett et al., 2012). Although the gut microbiome generally remains stable over long periods of time, its composition changes depending on external conditions such as changes in diet, antibiotic use, increased sanitation and stress. This leads to unbalanced gastrointestinal conditions called dysbiosis (Clemente et al., 2012). Dysbiosis is characterized by moderate or severe disturbances in the composition of the normal intestinal microbiome, which leads to the disappearance of key types of microorganisms, the loss of specific microbial functions and, as a consequence, disruption of the modulation of intestinal wall activity. This can lead to the colonization of pathogenic microorganisms and cause diarrhea or necrosing enteritis (Sekirov et al., 2008). One of the severe forms of this pathogenesis is CDAD ( Clostridium difficile associated diarrhea), the treatment of which with classical antibiotic therapy increasingly does not give the desired result. Other consequences of microbial dysbiosis may be an impaired immune response leading to chronic inflammation (Willing et al., 2009) or food allergies, or increased intestinal permeability, nutritional malabsorption or even bacteremia. The adverse effects of dysbiosis on microbial functionality and intestinal wall physiology can thus undermine human health. In fact, constipation, IBS, IBD, pouchitis, metabolic syndrome, obesity, diabetes, cardiovascular disease, psychiatric conditions, cognitive impairment, neurodegenerative diseases, various types of cancer (eg, colon cancer), inflammation of the female genital tract , CDAD, rheumatism or rheumatoid arthritis are associated with changes in the activity/composition of the gut microbiota. Therefore, it is quite obvious that dysbiosis must be prevented or eliminated when it appears.

If dysbiosis is associated with the presence of pathogens, then the obvious strategy used to remove harmful microorganisms is the use of antibiotics. However, in the last decade, widespread and misuse of broad-spectrum antibiotics has led to a dramatic increase in antibiotic resistance (Brandl et al., 2008). In addition, antibiotics also destroy natural intestinal microorganisms, many of which perform important functions and provide health benefits, that is, they prevent the development of dysbiosis. As a result, over the past 2 decades, a huge amount of research has been carried out on the search for beneficial foods, and in particular on the development of prebiotics and probiotics. Although the concept of prebiotics is attractive because it refers to nutritional modulation of natural intestinal microorganisms that have already adapted to the host (Van Loo et al., 1999), however, such a concept is used mainly for preventive purposes. For therapeutic applications, in the event of significant disruption of the gut microbiome, there would be greater benefit from introducing key microbial species rather than obtaining substrates that have beneficial effects on health-promoting species but which are less common or even absent in the affected individual. A possible solution to this problem is the introduction of viable, health-promoting microorganisms called probiotics (Lannitti & Palmieri, 2010). Probiotic products generally consist of 1 or 2 unrelated strains of microorganisms (mainly lactic acid-producing bacteria) with specific functionality. However, the survival of probiotic strains in the harsh conditions of the upper digestive tract is problematic, and there is little competition with the vast natural microbiome. However, the concept of introducing new species of microorganisms into the gut with a weakened ecosystem has been gaining momentum in recent years through the use of fecal microbial transplants (FMT) (Khoruts et al., 2010). This facilitates the transfer of fecal microbial suspension from a healthy donor to a sick recipient. This form of bacteriotherapy is used primarily to treat antibiotic-resistant infections and is effective in 90% or more of cases. FMT is currently used to treat many other pathologies that are caused by gastrointestinal dysbiosis (Crohn's disease, obesity, irritable bowel syndrome). It is believed that FMT can work effectively when there is a serious deficiency of single probiotic strains. However, the poorly characterized nature of fecal transplants has been associated with a risk of infectious disease transmission, and questions are currently being raised about their widespread use for less acute and life-threatening conditions (De Vrieze 2013).

In early 2013, an alternative to the use of fecal microbial transplants appeared in the publication of a scientific article (Petrof et al., 2013) and in the patent application WO2013037068: “Method for treatment of disorders of the gastrointestinal system” - (“Method for the treatment of disorders of the gastrointestinal tract” ), which describes the use of a synthetic mixture of microbes that have been isolated from humans based on their cultural properties and used as a therapeutic agent for the treatment of CDAD. Such a product also consists of a known series of microorganisms that would address problems associated with disease transmission from fecal transplants while meeting QPS criteria. However, mixing microorganisms with each other does not guarantee their interaction with each other and their filling of functional niches that require microbial networks. Therefore, product stability, standardization and performance of critical functions cannot be guaranteed.

Patent application WO2014145958A2 (Network-based microbial compositions and methods) proposes administering to a mammal in need of an effective amount of a therapeutic bacterial composition a plurality of isolated bacteria or a purified bacterial preparation. Many isolated bacteria or a purified bacterial preparation are capable of forming a so-called ecological network. For this drug, bacteria are selected based on genomic information and administered to the mammal as a set of strains belonging to distinct communities.

Becker et al. (2011) described a community consisting of 8 different strains: Anaerostipes caccae, Bacteroides thetaiotaomicron, Bifidobacterium longum, Blautia producta, Clostridium butyricum, Clostridium ramosum, Escherichia coli, Lactobacillus plantarum . This community is designated SIHUMIx (Simplified Human Microbiota Extended). This artificial community of microorganisms was tested in rat studies by comparing microorganisms in rats inoculated with SIHUMIx with microorganisms present in normal humans and uninfected rats. The authors argue that such a community is representative of the microbiota present in the human colon in terms of composition and functionality. These microbial communities evolved depending on the age of the rats, but over time, their composition became stable.

Van den Abbeele et al. (2013) suggested the possibility of generating glycan-degrading communities using conventional in vitro fermenters, which can be inoculated with appropriate key species and a mixture of microorganisms exhibiting symbiotic growth on minimal media. Once inoculated and stabilized, such a distinct community of microbial networks can be achieved that carry specific functions and are produced on a large scale.

Finally, Newton et al. (1998) used anaerobic chemostats to generate reproducibly defined bacterial communities including 14 different saccharolytic and amino acid-fermenting species (i.e., Bifidobacterium longum, Bif. adolescentis, Bif. pseudolongum, Bif. infantis, Bacteroides thetaiotaomicron, Bact. vulgatus, Lactobacillus acidophilus, Enterococcus faecium, Escherichia coli, Clostridium perfringens, Cl. butyricum, Cl. innocuum, Cl. bifermentans) to study the effect of sulfovibrio desulfuricans on other intestinal microorganisms.

However, there is still a need to develop alternative and specific mixtures of bacterial species that can be effectively used for the prevention or treatment of gastrointestinal disorders. Moreover, it is completely unknown whether pre-adapted mixtures, when administered, will have the same therapeutic properties as individual communities and pre-adapted mixtures of the same bacterial species, or whether they will have worse or better properties.

Brief description of drawings

Figure 1: Schematic representation of the SHIME® system, which consists of the stomach, small intestine and three different areas of the colon. SHIME® liquid culture medium and pancreatic somatic cells enter vessels that simulate the stomach and small intestine, respectively. After a certain residence time in these sterile vessels, the suspension occupies three successive compartments of the colon, that is, the ascending, transverse and descending colon, each characterized by a different pH and residence time. These vessels are seeded with human fecal microbiota. All vessels are stored under anaerobic conditions, created by purging the headspace zone with N ₂ , with continuous stirring and holding at 37°C.

Figure 2: Butyrate production by 23 different formulations after 24-hour incubation (top panel) and its effect on transepithelial electrical resistance (TEER) of Caco-2 cells cultured in the presence of THP1 cells (bottom panel). In the latter case, samples collected after a 24-hour incubation of 23 formulations were sterile filtered and added (1:5 v/v) for 24 hours to the apical compartment of Caco-2 cells cultured for 14 days on semi-permeable inserts and placed on the top of PMA-stimulated THP1-derived macrophages (co-cultures). Only one culture medium (DMEM) was used as a control. THP1 cells cultured in the presence of PMA for 48 hours caused damage to Caco-2 cells, as determined by a decrease in TEER in the DMEM control. TEER values were normalized to those measured before coculture (0 h) and expressed as a percentage of the original value. The various compositions were assigned the following codes: MX-Y, where X=number of isolates present in the composition and Y=unique composition A, B, C, etc. with isolates of X.

Figure 3: Butyrate production after 24-hour and 48-hour incubation in SHIME® conditioned culture medium with the complete 7-species composition or 6-species compositions, from which one of the 7 original species was excluded each time. Results are presented as the percentage of butyrate production detected in each incubation with the 6-species composition as opposed to the composition consisting of all 7 species. These compositions were designated as “Complete” (from all 7 species) or “complete - X”, where X is the species excluded from the complete composition. ^* p <0.05 compared to “Full composition” after 24 hours; # p <0.05 compared to “Full composition” after 48 hours.

Figure 4: Levels (mM) of butyrate, propionate and acetate produced by the composition during a 5-day anaerobic incubation in SHIME® conditioned culture medium. The composition was obtained using either a “microcommunity” strategy (left panel) or a “collaborome” strategy (right panel).

Figure 5: Change in levels (mM) of propionate (left panel) and butyrate (right panel) over 14 days in 3 independent formulation cycles using a “collaborome” strategy. After pre-cultivation in an appropriate culture medium, the strains of the composition were mixed, seeded and cultured for 14 days in triplicate in the SHIME® system, consisting of one area of the colon at pH 6.15-6.4.

Figure 6: Change in SCFA levels expressed as mol% acetate, propionate and butyrate over time after production of the composition using an alternative "colaborome" strategy. After pre-cultivation in an appropriate culture medium, the strains of the composition were mixed, seeded and cultivated for 8 days in triplicate in separate fermenters operating in fed-batch culture mode. At specified intervals over a period of 16 hours, 40% (by volume) of the culture medium was replaced with SHIME® conditioned growth medium.

Figure 7: Production (mM) of acetate, propionate, butyrate and total short chain fatty acids (SCFA) after 24 hours incubation with (i) sterile basal medium (top panel) or sterile medium supplied (ii) with microbiota, derived from the SHIME colon region (middle panel), or (iii) with fecal microbiota (bottom panel). Various treatments of the composition produced using the "collaborome" strategy were carried out ranging from 0% to 4% and 20% of the total incubation volume.

Figure 8: Change in levels (mM) of acetate (top panel), propionate (middle panel) and butyrate (bottom panel) in the M-SHIME® antibiotic loading experiment. After inducing dysbiosis of the colon microbiota in the SHIME® system by administering a cocktail of antibiotics (40/40/10 mg/L amoxicillin/ciprofloxacin/tetracycline, respectively), the dysbiosis microbiota was treated for 5 days with a composition obtained using either strategy “microcommunities”, or using the “collaborations” strategy (day 1=beginning of the introduction of the composition). Results are expressed as delta SCFA levels in SHIME at each time point compared to pre-antibiotic values.

Figure 9: Levels (mM) of acetate (top panel), propionate (middle panel) and butyrate (bottom panel) in the IBD-associated dysbiosis reversal experiment in M-SHIME®. Three independent SHIME® colonic vials were inoculated with fecal material collected from a patient with ulcerative colitis. At the same time, a single dose of the composition obtained using either the “microcommunity” strategy or the “collaborome” strategy was administered into the corresponding SHIME colon vessel. The third experiment was carried out in parallel as a control without the introduction of the composition. The production of acetate, propionate and butyrate was observed 1 and 2 days after administration of the composition.

Figure 10: Changes in the levels (mol %) of acetate, propionate and butyrate in the experiment on the administration of antibiotics to C57/BL6 mice. After a control period during which mice were fed standard chow, gut microbiota dysbiosis was induced by adding clindamycin (250 mg/L) to drinking water for 5 days. Thereafter, mice (n=10/group) were administered orally by gavage for 5 days with either saline (no bacteria control; left panel) or a formulation prepared using the collaboration strategy (middle panel). or a composition with an expanded composition obtained using the “collaborative” strategy (right panel). Mice fecal samples collected from the same experimental group were pooled and levels of acetate, propionate and butyrate were quantified.

Figure 11: Change in disease severity index (DAI) and weight change in the TNBS-induced colitis experiment in C57/BL6 mice. After a 1-week acclimatization period, in which the mice were given standard food, the experiment began. Animals in each group (n=9/group) were treated for 5 consecutive days using an oral gavage. For all treatments, a prophylactic dose was administered 1 day before rectal administration of 2 mg TNBS/50% EtOH, and the experiment was completed 4 days after TNBS administration, and then the mice were sacrificed. The following treatments were carried out: TNBS+saline (control TNBS as a carrier); TNBS+composition obtained using the “microcommunities” strategy, and TNBS+composition obtained using the “collaborome” strategy. The normal group (without TNBS treatment but with saline treatment) was included as a vehicle control.

Figure 12: Changes in disease severity index (DAI) were observed in the DSS-induced colitis experiment in C57/BL6 mice. After a 1-week acclimatization period, in which the mice were given standard food, the experiment began. Animals in each group (n=10/group) were treated 3 times a week for 8 consecutive days using an oral gavage. For all treatments, the prophylactic dose was administered 1 week before the first DSS cycle. The first DSS cycle started from week 2, and this cycle included the administration of DSS once a week (0.25% in drinking water) followed by a break of two weeks. After the first cycle, an identical second DSS cycle was performed. The third DSS cycle consisted of DSS administration once a week followed by a one-week break, after which the animals were sacrificed. In this case, the following treatments were carried out: DSS + saline (control DSS as a carrier); DSS+composition obtained using the “micro-community” strategy. The normal group (without DSS treatment but with saline treatment) was included as a vehicle control.

The essence of the invention

The present invention relates primarily to a composition consisting essentially of bacteria belonging to the species Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburia inulinivorans, Roseburia hominis, Akkermansia muciniphila, Lactobacillus plantarum and Anaerostipes caccae, preferably used for the prevention or treatment of symptoms associated with gastrointestinal distress.

In other words, the present invention relates to a method of preventing or treating symptoms associated with a gastrointestinal disorder in an individual in need thereof, wherein said method comprises administering a therapeutically effective amount of a composition substantially consisting of bacteria belonging to the species Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburia inulinivorans, Roseburia hominis, Akkermansia muciniphila, Lactobacillus plantarum and Anaerostipes caccae .

The present invention also relates to the composition described above, wherein said gastrointestinal disorder is intestinal barrier dysfunction, diarrhea, constipation, irritable bowel syndrome, inflammatory bowel disease, Crohn's disease, ulcerative colitis, celiac disease, pouchitis, mucositis, infection intestines, dysbiosis of the intestinal microbiota, or any combination thereof.

The present invention also relates to the composition described above, wherein said gastrointestinal disorder is prevented or treated by: a) stimulating the growth and/or activity of one or a limited number of beneficial bacteria in the intestinal tract, b) inhibiting the growth and/or activity of one or a limited number of beneficial bacteria the number of pathogenic bacteria in the intestinal tract, c) a relative increase in the level of binding of non-pathogenic bacteria to the mucous membrane of the gastrointestinal surface, d) a decrease in the uncontrolled absorption of antigens, pro-inflammatory agents, bacteria or bacterial products by the intestine, e) providing anti-inflammatory activity at the intestinal surface, f) improving the functioning of the intestinal barrier, g) obtaining bacterial metabolites or h) any combination of a)-g).

The present invention also relates to the composition described above, wherein a bacterium belonging to the species Roseburia hominis has been removed from the composition.

The present invention also relates to the composition described above, wherein a bacterium belonging to the species Escherichia coli, Enterococcus faecium, Lactobacillus mucosae, Bifidobacterium adolescentis, Bifidobacterium longum, Bacteroides thetaiotaomicron and Bacteroides vulgatus has been further added to the said composition.

The present invention also relates to the composition described above, which also contains one or more prebiotics.

In its preferred embodiment, the present invention relates to the composition described above, wherein said bacteria are pre-adapted by co-cultivating them in a fermenter before administering said composition for the prevention or treatment of said gastrointestinal disorders.

Accordingly, the present invention also relates to the composition described above, wherein said fermenter is a dynamic gastrointestinal tract simulator.

More specifically, the present invention relates to the composition described above, where the said bacteria are selected from the list of the following strains: Faecalibacterium prausnitzii LMG P-29362, Faecalibacterium prausnitzii DSMZ 17677, Butyricicoccus pullicaecorum LMG P-29360, Butyricicoccus pullicaecorum LMG24109, Roseburia inulinivorans LMG 65, Roseburia inulinivorans DSMZ 16841, Roseburia hominis LMG P-29364, Roseburia hominis DSMZ 16839, Akkermansia muciniphila LMG P-29361, Akkermansia muciniphila DSMZ 22959, Lactobacillus plantarum LMG P-29366, Lactobacillus plantarum ZJ31 6, Anaerostipes caccae LMG P-29359, Anaerostipes caccae DSMZ 14662 and/or strains whose sequences are at least 97% identical to the 16S rRNA sequences of at least one of these strains.

The present invention also relates to the composition described above, wherein said composition is a pharmaceutical composition formulated as a dosage form for rectal administration or as a dosage form for oral administration.

Accordingly, the present invention also relates to the composition described above, wherein said oral dosage form is a capsule, microcapsule, tablet, granule, powder, lozenge, pill, suspension or syrup.

The present invention also relates to the composition described above which is included in foods, beverages, dietary supplements or natural preparations.

More specifically, the present invention relates to the composition described above, wherein the composition contains from 10 ⁵ to 10 ¹¹ colony forming units of bacteria.

Description of the invention

The gut microbiome contains hundreds of species of microorganisms that coexist in different individuals and that interact with each other and with the host. Currently, it is generally believed that the gut microbiota plays a key role in human health and disease prevention through the regulation of metabolic functions and immune homeostasis (Cenit et al., 2014). Several studies have been conducted to examine complex gut microbial communities in an attempt to define a “core microbiome,” meaning that each individual shares a key number of core species or strains that determine the functionality of a healthy gut microbiome (Kinross et al., 2011). Based on this concept (i.e., that all humans are inhabited by a core microbiome), there is an extensive literature describing the composition and function of the gut microbiota (e.g., key bacterial species, mucosal and luminal microbiota, proximal and distal colonic bacteria, etc. .) and functional genome analysis, and which provides a comprehensive list of candidate microbes covering the core functional properties of the complex human gut microbiome.

The present invention relates primarily to a specific selection of a subset of bacterial species in the human gut microbiome that produces a specific and unexpected effect. More specifically, the present invention relates to a composition consisting essentially of bacteria belonging to the species Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburia inulinivorans, Roseburia hominis, Akkermansia muciniphila, Lactobacillus plantarum and Anaerostipes caccae , which are preferably used for the prevention or treatment of symptoms, associated with gastrointestinal distress. The term "consisting essentially of" indicates that the composition may include other species of bacteria and/or other components, provided that they do not adversely affect the effect (i.e., the prevention or treatment of symptoms associated with gastrointestinal tract). intestinal disorder) of the said composition. In one embodiment of the invention, the composition according to the invention includes bacteria belonging to the species Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburia inulinivorans, Roseburia hominis, Akkermansia muciniphila, Lactobacillus plantarum and Anaerostipes caccae .

In another embodiment of the invention, the composition according to the invention consists of bacteria belonging to the species Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburia inulinivorans, Roseburia hominis, Akkermansia muciniphila, Lactobacillus plantarum and Anaerostipes caccae .

Bacteria of the species Faecalibacterium prausnitzii (Duncan et al., 2002), Butyricicoccus pullicaecorum (Eeckhaut et al., 2008), Roseburia inulinivorans (Duncan et al., 2006), Roseburia hominis (Duncan et al., 2006), Akkermansia muciniphila (Derrien et al., 2004), Lactobacillus plantarum (Walter, 2008) and Anaerostipes caccae (Schwiertz et al., 2002) are bacteria well known to those skilled in the art. The term "symptoms associated with gastrointestinal distress" refers to health problems in humans and animals. The use of the composition according to the invention allows, in particular, the prevention/treatment of dysbiosis, and thereby contributes to the positive modulation of interactions between bacteria and the intestinal surface. The result is improved intestinal surface functioning, such as improved barrier, hormonal and immune function. The effect on the intestinal surface occurs more quickly when a dose of a “pre-adapted composition” is administered compared to a dose of a series of the same strains separately (see below). As used herein, the term "modulation or improvement of the barrier, hormonal and immune functions of the intestinal surface" means the modification of any parameter that affects the normal homeostasis of the intestinal surface, and in particular, its role in the first line defense against invasion of pathogens, antigens or other harmful substances, and its role in the production of substances (eg, immune molecules, hormones) that have systemic effects on the host. These options include, but are not limited to:

- stimulation of the growth and/or activity of one or a limited number of beneficial bacteria in the gastrointestinal tract (for example, lactobacilli, bifidobacteria, butyrate- or propionate-producing bacteria, etc.);

- inhibition of the growth and/or activity of one or a number of pathogenic bacteria in the intestinal tract;

- a relative increase in the attachment of non-pathogenic bacteria to the mucous membrane of the intestinal surface;

- reducing the uncontrolled absorption of antigens, pro-inflammatory molecules, bacteria or bacterial products by the intestines;

- modulation of intestinal lymphoid tissue (GALT) and the host immune system;

- production of specific bacterial metabolites (for example, propionate, butyrate); and modulation of the production of certain intestinal signaling molecules that directly or indirectly modulate metabolic homeostasis (eg, proglucagon, GLP-1, GLP-2, FIAF).

Thus, the present invention relates to a composition as described above, wherein said gastrointestinal disorder is prevented or treated by: a) stimulating the growth and/or activity of one or a limited number of beneficial bacteria in the intestinal tract, b) inhibiting the growth and/or activity one or a limited number of pathogenic bacteria in the intestinal tract, c) a relative increase in the level of binding of non-pathogenic bacteria to the mucosal surface of the gastrointestinal surface, d) a decrease in the uncontrolled absorption of antigens, pro-inflammatory agents, bacteria or bacterial products by the intestine, e) providing anti-inflammatory activity at the intestinal surface , f) improving the functioning of the intestinal barrier, g) obtaining bacterial metabolites, or h) any combination of a)-g).

Conditions that may be associated with common gastrointestinal disorders include, but are not limited to, constipation, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), gut microbiota dysbiosis, mucosal inflammation, metabolic syndrome, obesity, diabetes, cardiovascular disease, chronic fatigue syndrome, mental illness, cognitive impairment, neurodegenerative disease, cancer of any form, autoimmune disease, immune system dysfunction, rheumatism, rheumatoid arthritis, inflammation of the female reproductive organs, infection by pathogens (bacteria, viruses and mushrooms). Examples of neurodegenerative diseases include, but are not limited to, ABS, dementia, Alzheimer's disease, Parkinson's disease and Huntington's disease. Examples of various types of cancer include, but are not limited to, lung cancer, breast cancer, prostate cancer, pancreatic cancer, and in particular, colorectal cancer. Examples of autoimmune diseases include, but are not limited to, multiple sclerosis, atopic dermatitis, celiac disease, psoriasis, and lupus.

Based on the observation that the compositions of the invention enhance the interaction and/or activity of non-pathogenic bacteria on the mucosal lining of the gastrointestinal epithelium, it is believed that these preparations are particularly suitable for improving the barrier function of the intestinal surface, such as, for example, preventing or reducing uncontrolled absorption antigens, pro-inflammatory molecules, pathogenic bacteria or bacterial products of the intestine. One such sign of impaired mucosal barrier function is inflammatory bowel disease. Since it is generally believed that in inflammatory bowel diseases, damage to the mucosa with impaired recovery from such damage is one of the key factors causing such chronic diseases, the compositions of the invention should have a beneficial effect on this condition. Accordingly, it is an object of the present invention to use the compositions of the invention for the prevention and treatment of conditions associated with impaired barrier function and characterized by uncontrolled uptake of antigens, proinflammatory molecules, pathogenic bacteria or bacterial products by the intestine.

As used herein, the term "inflammatory bowel diseases" is also referred to as "chronic intestinal diseases", and this term includes any condition characterized by persistent inflammation of the mucous membrane at various levels of the gastrointestinal tract, such as, for example, inflammatory bowel syndrome, mucosal inflammation, stomach ulcers, Crohn's disease, ulcerative colitis, rectal and colon cancer and pouchitis.

Since it is well known that mucosal inflammation is mainly characterized by inflammation of the surface lining of the oral mucosa and gastrointestinal tract, which is usually a side effect of chemotherapy and radiation therapy or stem cell transplantation, it should also be noted that the use of the compositions according to the invention will have a beneficial effect in such conditions. Accordingly, it is an object of the present invention to use the compositions according to the invention for the prevention and treatment of conditions associated with inflammation of the mucous membrane. Inflammation of the mucous membrane can occur in any area throughout the gastrointestinal tract. In the case of the oral cavity, it is usually called inflammation of the oral mucosa.

It should also be noted that the use of the compositions according to the invention provides protection against the invasion of antigens that cause allergic reactions, since such allergens may be contained in certain foods, chemicals and other molecules. Thus, in another embodiment, the present invention relates to the use of compositions for the prevention and treatment of conditions associated with invasion of antigens that cause allergic reactions (eg, food allergies, asthma, eczema).

In addition, it should be noted that the use of these compositions affects the intestinal lymphoid tissue (GALT) as well as the immune system. Among other effects, this may lead to decreased expression of proinflammatory cytokines and increased production of immunoregulatory factors, as well as increased lymphocyte activity. Therefore, it is believed that these compositions are particularly suitable for improving the development and functioning of the host immune system.

In another aspect of the invention, based on the observation that the compositions of the invention modulate the epithelial barrier, and therefore reduce chronic inflammation, it is believed that the compositions are particularly suitable for regulating and improving metabolic homeostasis. Non-limiting effects of these drugs on metabolic homeostasis include regulation of food digestibility and metabolism of fat and glucose, increased insulin secretion and insulin sensitivity, and regulation of cholesterol synthesis and metabolism. Accordingly, the purpose of the present invention is the use of the compositions according to the invention for regulating food digestibility, stimulating the feeling of satiety, regulating body weight, preventing and treating conditions associated with disturbances of metabolic homeostasis, such as obesity and type 2 diabetes.

Based on the observation that the composition of the invention reduces certain established causative risk factors for the development of cardiovascular disease (CVD), it is believed that in another aspect of the invention, these compositions are particularly suitable for the prevention of CVD. In fact, CVD refers to any disease that affects the cardiovascular system, but usually the term refers to disease associated with atherosclerosis. Atherosclerosis is a syndrome of arterial blood vessel disease and chronic inflammatory reactions in the artery walls, which is largely caused by the accumulation of macrophages, white blood cells and low-density lipoproteins. The development of CVD depends on multiple mechanisms, and a number of clear causative risk factors have also been identified. These factors include, but are not limited to, elevated LDL cholesterol, elevated plasma triglycerides, metabolic diseases (obesity, diabetes, etc.), chronic inflammation and oxidative stress. The last two factors are especially important. Atherosclerosis develops as a result of the oxidation of LDL (LDL-ox) by free radicals, and in particular by oxygen free radicals in the case of oxidative stress. An overreaction of the immune system, in the case of chronic inflammation, leads to damage caused by LDL oxidation, which also contributes to increased disease severity. Therefore, the purpose of the present invention is the use of the compositions according to the invention for the prevention or treatment of CVD.

In another aspect of the invention, given the beneficial effect of the compositions of the invention on the adhesion of normal microbiota to the mucosal layer, it was hypothesized that the use of the compositions would provide protection against mucosal adhesion and pathogen invasion. Examples of pathogens include, but are not limited to, Bacillus anthracis; Bacillus cereus; Bordetella pertussis; Borrelia burgdorferi; Brucella abortus; Brucella canis; Brucella melitensis; Brucella suis; Campylobacter jejuni; Chlamydia pneumonia; Chlamydia trachomatis; Chlamydophila psittaci; Clostridium botulinum; Clostridium difficile; Clostridium perfringens; Clostridium tetani; Corynebacterium diphtheria; enterotoxigenic Escherichia coli (ETEC); enteropathogenic E. coli; E. coli 0157.Ή7; Francisella tularensis; Haemophilus influenza; Helicobacter pylori; Legionella pneumophila; Leptospira interrogans; Listeria monocytogenes; Mycobacterium leprae; Mycobacterium tuberculosis; Mycoplasma pneumonia; Neisseria gonorrhoeae; Neisseria meningitides; Pseudomonas aeruginosa; Rickettsia rickettsia; Salmonella typhi; Salmonella typhimurium; Shigella sonnei; Staphylococcus aureus; Staphylococcus epidermidis; Staphylococcus saprophyticus; Streptococcus agalactiae; Streptococcus pneumonia; Streptococcus pyogenes; Treponema pallidum; Vibrio cholera; Yersinia pestis; Candida spp.; norovirus (Norwalk virus); hepatitis A virus; viruses that cause smallpox, influenza, mumps, measles, chicken pox; Ebola virus and rubella measles virus. Thus, in another embodiment, the present invention relates to the use of the compositions according to the invention for the prevention and treatment of conditions associated with mucosal adhesion and invasion of pathogens, and in particular for the treatment and prevention of acquired diarrhea and traveler's diarrhea.

The present invention also relates to a method of preventing or treating symptoms associated with a gastrointestinal disorder in an individual in need thereof, wherein said method comprises administering a therapeutically effective amount of a composition consisting primarily of bacteria belonging to the species Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum , Roseburia inulinivorans, Roseburia hominis, Akkermansia muciniphila, Lactobacillus plantarum and Anaerostipes caccae .

The term “individual in need of treatment” refers to a person or non-human animal having a gastrointestinal disorder as described above.

The term "therapeutically effective amount" means the minimum combined total amount of bacteria of 7 species that are capable of producing a prophylactic or therapeutic effect. These 7 bacterial species are listed below: Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburia inulinivorans, Roseburia hominis, Akkermansia muciniphila, Lactobacillus plantarum and Anaerostipes caccae .

However, the term "therapeutically effective amount" may also mean the minimum combined total number of bacteria of the 6 species listed below: Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburia inulinivorans, Akkermansia muciniphila, Lactobacillus plantarum and Anaerostipes caccae .

Depending on the purpose of use, the specified combined total amount may be the sum of equal amounts of each of the 7 bacterial species or unequal amounts of each of the 7 bacterial species, where each one of the 7 bacterial species has a minimum excess of 0.0001% of the combined total, more preferably, a minimum excess of 0.001% of the combined total, and most preferably, a minimum excess of 0.01% of the combined total. Thus, if, for example, 6 species of bacteria have an excess of 10.00% of the combined total, then the 7th species has an excess of 40.00% of the combined total. Depending on the purpose of use, said aggregate total amount is within a daily dosage range of 10 ² to 10 ¹⁴ bacterial cells, preferably within a daily dosage range of 10 ³ to 10 ¹³ bacterial cells, more preferably within a daily dosage range of 10 ⁴ to 10 ¹² bacterial cells, and most preferably within a daily dose of 10 ⁵ to 10 ¹¹ bacterial cells.

The present invention also relates to the composition described above, from which bacteria belonging to the species Roseburia hominis are excluded. The term "excluded" refers specifically to the preparation of a composition of 6 species of bacteria, as described below in the Examples section, without adding or removing Roseburia hominis bacteria as a 7th species bacteria.

The present invention also relates to the composition described above to which bacteria belonging to the species Escherichia coli, Enterococcus faecium, Lactobacillus mucosae, Bifidobacterium adolescentis, Bifidobacterium longum, Bacteroides thetaiotaomicron and Bacteroides vulgatus have been added.

Bacteria species Escherichia coli (Rath et al. 1999), Enterococcus faecium (Schleifer et al. 1984), Lactobacillus mucosae (Roos et al. 2000), Bifidobacterium adolescentis (Scharek et al. 2000), Bifidobacterium longum (Bahaka et al. 1993 ), Bacteroides thetaiotaomicron (Scharek et al. 2000) and Bacteroides vulgatus (Rath et al. 1999) are well known to those skilled in the art. The present invention also relates to the composition described above, which also contains one or more prebiotics.

The term "prebiotic" means any chemical substance that induces the growth or activity of microorganisms (eg, bacteria) that have a beneficial effect on the host. Therefore, prebiotics can influence or change the composition of microorganisms in the gut microbiome. However, in principle, the term has a more general meaning that can also apply to other areas of the body. Exemplary, but not limiting, prebiotics are non-fiber-degrading compounds that at least partially pass through the upper gastrointestinal tract in an unhydrolyzed form and stimulate the growth or activity of beneficial bacteria that colonize the colon through their substrate action.

In its preferred embodiment, the present invention relates to the composition described above, wherein said bacteria are cultured together in a fermenter, and then said composition is administered for the prevention or treatment of said gastrointestinal disorders. These compositions are also referred to (see below) as "collaborom strategy" or "alternative collaborom strategy" compositions. In contrast, a composition where said bacteria are not cultured together in a fermenter prior to their introduction is called a composition (see below) obtained using a “micro-community strategy”.

Accordingly, the present invention also relates to the composition described above, wherein said fermenter is a dynamic gastrointestinal tract simulator. In this particular case, the latest compositions are also called (see below) as a “collaboration strategy”.

SHIME® (Simulator of the Human Microbial Ecosystem) is a dynamic in vitro model of the human gastrointestinal tract that consists of five double-jacketed vessels simulating the stomach, small intestine, and three regions of the colon (ascending, transverse, and descending colon) , with a total retention time of 72 hours (Fig. 1). 140 ml of SHIME® nutrient medium and 60 ml of pancreatic juice were added to mock stomach and small intestinal vessels three times a day, respectively (Van den Abbeele et al., 2010). After an initial 2-week stabilization period, which allows the microbiota to adapt to the established in vitro conditions, the isolation procedure was started. Thus, selected microbial strains according to the invention can be inoculated into single-stage reactors (alternative collaboration strategy) or into multi-stage reactors or dynamic gastrointestinal simulators (e.g. SHIME® or M-SHIME®, collaboration strategy) under standard conditions that are representative of the gastrointestinal tract. Accordingly, the present invention relates to a reactor containing a composition comprising, consisting of, or essentially consisting of bacteria belonging to the 6 or 7 to 14 species defined herein and listed below, wherein said reactor :

- includes a composition containing, consisting of or essentially consisting of bacteria belonging to the species Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburia inulinivorans, Roseburia hominis, Akkermansia muciniphila, Lactobacillus plantarum and Anaerostipes caccae , or

- includes a composition containing, consisting of or essentially consisting of bacteria belonging to the species Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburia inulinivorans, Akkermansia muciniphila, Lactobacillus plantarum and Anaerostipes caccae or

- includes a composition containing, consisting of or essentially consisting of bacteria belonging to the species Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburia inulinivorans, Roseburia hominis, Akkermansia muciniphila, Lactobacillus plantarum, Anaerostipes caccae, Escherichia coli, Enterococcus faecium, Lactobacillus mucosae, Bifidobacterium adolescentis, Bifidobacterium longum, Bacteroides thetaiotaomicron and Bacteroides vulgatus .

In a preferred embodiment of the invention, this reactor containing said composition is operated under standardized conditions representative of the gastrointestinal tract, as defined below.

Parameters characterizing standardized conditions include, but are not limited to: pH (ranging from 1.5 to 8); presence of carbon sources (carbohydrates or proteins or combinations thereof); retention time in a particular reactor (ranging from 10 minutes to 200 hours); oxygen availability (ranging from 0 to 8 g/l); presence of microelements; presence/absence of antibiotics; concentration of bile salts (ranging from 0 to 20 mM); presence of heavy metals; the presence of factors such as host immune system molecules. In a preferred embodiment of the invention, parameters characterizing the standardized conditions include pH, retention time in a particular reactor, and concentration of bile salts, as defined above. Depending on the complexity of the collaborome, a period of time ranging from 1 to 15 days is required to obtain a functionally stable collaborome. On average, for collaboromes consisting of 7-14 members, the time required to obtain a functionally stable collaborome ranges from 3 to 10 days (depending on environmental conditions). Therefore, the composition as defined herein can be prepared after "forming" or culturing the bacteria for a period of time from 3 to 10 days under conditions where the pH, retention time in a particular reactor, and concentration of bile salts have been determined herein. This method makes it possible to obtain a composition or collaboration that is functionally stable.

In the context of the present invention, the term “functionally stable collaborom” means a composition as defined herein containing a different initial number of bacterial species after cultivation for at least 3 or 5 or 10 days.

In another aspect, the present invention relates to a reactor operating under standardized conditions representative of the gastrointestinal tract, wherein said reactor has the following parameters: pH ranging from 1.5 to 8; availability of carbon sources; retention time from 10 minutes to 200 hours; oxygen availability ranging from 0 to 8 g/l; presence of microelements; presence/absence of antibiotics; concentration of bile salts ranging from 0 to 20 mM; presence of heavy metals; the presence of factors such as host immune system molecules. In one embodiment of the invention, the specified reactor has parameters characterizing standardized conditions, including pH, retention time in a particular reactor and concentration of bile salts, which are defined in the previous paragraph. In one embodiment of the invention, said reactor contains a composition of 5-20 bacteria of different species, or 6-14 bacteria of different species, or 5-15 bacteria of different species. In a preferred embodiment of the invention, the retention time of such a composition in the reactor is from 3 to 14 days or from 3 to 10 days, which allows to obtain a functionally stable collaborom.

More specifically, the present invention relates to the composition and use of a set of microbial strains having specific functional properties and previously adapted to function together, for the purpose of preventing or treating diseases in humans and animals and achieving faster biotherapeutic action and higher efficiency compared to those the same strains in minimal communities (“microcommunity strategy”). Such a set of microorganisms, previously adapted for their joint functioning, is called a “collaborome strategy” or “alternative collaborome strategy.”

In other words, the present invention relates to pre-adapted compositions consisting of a series of microbial strains, preferably used for the purpose of significantly reducing the time to onset of biotherapeutic effect and/or significantly increasing the effectiveness of treating dysbiosis compared to the same microbial strains in minimal communities.

The term "significant reduction in time to onset of biotherapeutic effect" means that a series of microorganisms, after their preliminary adaptation, can function at least 5% faster (on a time scale), preferably at least 10% faster, more preferably less 20% faster, and most preferably at least 30% faster, compared to recruiting the same strains in minimal communities. Any value below 5% is considered physiologically insignificant.

The terms "significantly improved treatment effectiveness" means that the series of microorganisms, after being previously adapted, can function at least 5% more effectively, preferably at least 10% more effectively, more preferably at least 20% more effectively, and most preferably at least 30% more effective. The effectiveness depends on the ultimate purpose of using the series of microorganisms. Possible functional parameters include, but are not limited to, production of short chain fatty acids (SCFA); improving intestinal barrier permeability; decrease/increase in the level of pro-inflammatory cytokines; increased levels of anti-inflammatory cytokines; reduction in pathogen concentrations (at least 0.5 log); reduction of gas emission; stimulation of specific receptors of the intestinal wall; and so on. Any value below 5% is considered physiologically insignificant.

Therefore, more specifically, the present invention relates to a method for preventing or treating dysbiosis in humans and animals in need thereof, wherein said method comprises administering a therapeutic amount of a pre-adapted composition of a series of microbial strains to said human or animal, wherein said treatment significantly reduces the time to onset of biotherapeutic effect and/or significantly increase the effectiveness of treatment compared to the introduction of a series of the same microbial strains in minimal communities.

More specifically, the present invention relates to the composition described above, in which the specified bacteria are selected from the list of the following strains: Faecalibacterium prausnitzii LMG P-29362, Faecalibacterium prausnitzii DSMZ 17677, Butyricicoccus pullicaecorum LMG P-29360, Butyricicoccus pullicaecorum LMG24109, Roseburia inulinivorans LMG 365 , Roseburia inulinivorans DSMZ 16841, Roseburia hominis LMG P-29364, Roseburia hominis DSMZ 16839, Akkermansia muciniphila LMG P-29361, Akkermansia muciniphila DSMZ 22959, Lactobacillus plantarum LMG P-29366, Lactobacillus plantarum ZJ31 6, Anaerostipes caccae LMG P-29359, Anaerostipes caccae DSMZ 14662 and/or strains whose sequences are at least 97% identical to the 16S rRNA sequences of at least one of these strains.

The above strains, having registration numbers LMG P-29362, LMG P-29360, LMG P-29365, LMG P-29364, LMG P-29361, LMG P-29366 and LMG P-29359, were deposited in the BCCM/LMG Laboratorium voor Microbiologie, Universiteit Gent (UGent), K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium.

The above strains, with registration numbers DSMZ 17677, LMG24109, DSMZ 1 6841, DSMZ 16839, DSMZ 22959, ZJ316 and DSMZ 14662, have been described in detail and deposited in public collections and are available to qualified specialists around the world.

It is also clear that variants of each of these strains that have a sequence that is at least 97% (ie, 97, 98, 99%) homologous to the 16S rRNA sequence of each of these respective strains are also part of the present invention. For example, the definition of “homology” of such a sequence is given, for example, by Eeckhaut et al. (2008). As used herein, the term “16S rRNA” refers to a nucleic acid sequence of approximately 1542 nucleotides that is a component of the small prokaryotic ribosomal subunit (30S). It is known that 16S rRNA acts as a scaffold that determines the positions of ribosomal proteins. The 16S rRNA sequence is commonly used for phylogenetic studies because it is known to be highly conserved. Comparative analysis of 16S rRNA sequences from thousands of microorganisms indicated the presence of signature oligonucleotide sequences. As used herein, the term “homology” means similarity of nucleic acid sequences. So, for example, in general, if two nucleic acids have identical sequences, this means that they have 100% homology. A change in the nucleotide sequence of one of the nucleic acids leads to a decrease in the percentage of homology. Generally speaking, percent homology quantifies the degree of identity between two nucleic acid sequences.

Sequence identity or sequence homology is defined herein as the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid sequences (polynucleotide sequences) as determined by sequence comparison. Typically, sequence identity or similarity is determined over the entire length of the sequences being compared. In the literature, the term "identity" is also defined as the degree of relatedness of amino acid sequences or nucleic acid sequences, and depending on the situation, it can be determined by the similarity of fragments of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutions in one polypeptide with the sequence of a second polypeptide. “Identity” and “similarity” can be easily calculated in various ways known to those skilled in the art. In a preferred embodiment of the invention, sequence identity is determined by comparing the sequences along their entire length, as defined herein.

Preferred identity determination methods have been developed to determine the best match between test sequences. Methods for determining identity and similarity were represented by codes in publicly available computer programs. Preferred computer programs for determining the identity and similarity of two sequences are, for example, BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990), and these programs are available in the NCBI public database and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894) The most preferred algorithm used is EMBOSS (httpi//www.ebi.ac.uk/emboss/). aliqn). The preferred parameters for comparing amino acid sequences using EMBOSS are gap-to-gap 10.0, gap-to-extend 0.5, Blosum matrix 62. The preferred parameters to compare nucleic acid sequences using EMBOSS are gap-to-gap 10.0, space. elongation 0.5, full-length DNA template (matrix for determining DNA identity).

When determining the degree of similarity of amino acid sequences, one skilled in the art may also, but not necessarily, consider so-called “conservative” amino acid substitutions that would be obvious to those skilled in the art. Conservative amino acid substitutions mean interchangeable residues that have similar side chains. For example, the group of amino acids with aliphatic side chains includes glycine, alanine, valine, leucine and isoleucine; the group of amino acids having aliphatic hydroxyl side chains includes serine and threonine; the group of amino acids having amide-containing side chains includes asparagine and glutamine; the group of amino acids with aromatic side chains includes phenylalanine, tyrosine and tryptophan; the group of amino acids with main side chains includes lysine, arginine and histidine; and the group of amino acids that have sulfur-containing side chains includes cysteine and methionine. Preferred conservative groups of amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine and asparagine-glutamine. Variants of the amino acid sequences described herein with substitutions are sequences in which at least one residue in the described sequences has been deleted and another residue has been introduced in its place. Preferably, the amino acid substitution is conservative. The preferred conservative substitutions for each naturally occurring amino acid are: Ala to Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser or Ala; Gln to Asn; Glu to Asp; Gly on Pro; His to Asn or Gln; Ile to Leu or Val; Leu on Ile or Val; Lys to Arg, Gln or Glu; Met on Leu or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and Val on Ile or Leu.

Those skilled in the art are well aware that 16s rRNA sequences can be deposited online, for example in GenBank (http://www.ncbi.nlm.nih.gov/genbank/), and that they can be retrieved by their unique accession number for use as a reference 16s rRNA sequence when assessing sequence homology, as described, for example, by Eeckhaut et al. (2008). GenBank accession numbers for 16s rRNA sequences of compositions from 7 bacterial species are listed below. To obtain the corresponding 16s rRNA sequences, these accession numbers can be taken from http//www.ncbi.nlm.nih.gov/qenbank/ to assess sequence homology.

View Strain GenBank registration number
(http//www.ncbi.nlm.nih.gov/qenbank/) Roseburia hominis DSMZ 16839 AJ270482.2 (SEQ ID NO:1) Roseburia inulinivorans DSMZ 16841 AJ270473.3 (SEQ ID NO:2) Akkermansia muciniphila DSMZ 22959 AY271254.1 (SEQ ID NO:3) Anaerostipes caccae DSMZ 14662 AJ270487.2 (SEQ ID NO:4) Faecalibacterium prausnitzii DSMZ 17677 AJ270469.2 (SEQ ID NO:5) Lactobacillus plantarum ZJ316 JN126052.2 (SEQ ID NO:6) Butyricicoccus pullicaecorum LMG 24109 HH793440.1 (SEQ ID NO:7)

The present invention also relates to the composition described above, wherein said composition is a pharmaceutical composition prepared either as a dosage form for rectal administration or for oral administration.

Accordingly, the present invention also relates to the composition described above, wherein said oral dosage form is a capsule, microcapsule, tablet, granule, powder, lozenge, pill, suspension or syrup.

The present invention also relates to the composition described above which is included in foods, beverages, dietary supplements or natural preparations.

Thus, the present invention also relates to the composition described above, which is used as a food product, dietary supplement or drug for the treatment of humans, domestic animals, farmed animals or aquatic animals. Thus, the composition can be incorporated into a food product, functional foods, dietary supplements, cosmetics, natural preparations, probiotic composition or pharmaceutical preparation. The food product is typically an edible product consisting primarily of one or more proteins, carbohydrates and fats containing macronutrients. The food product may also contain one or more micronutrients such as vitamins or minerals.

As used herein, the term "food product" also includes beverages. Examples of food products in which such a composition may be included are snack pies, cereals, buns, muffins, cookies, cakes, confectionery products, processed vegetables, sweets, probiotic preparations, including yoghurts, drinks, liquids based on vegetable oils , liquids based on animal fat, frozen confectionery and cheeses. Preferred foods are yoghurts, cheeses and other dairy products. Examples of beverages include soft drinks, syrups, squash (juice with pulp), powdered drink mixes, and nutritional drinks. A natural product is a food ingredient, food additive or food product that is believed to provide health benefits, including the prevention and treatment of disease. A functional food is a food product that is typically marketed as a wholesome, neat food product for post-meal consumption.

The present invention also relates to a probiotic containing the composition discussed herein. A probiotic is usually a “live” supplement that can improve the gut microbiota. Such probiotics can in particular be formulated for humans, but also for agricultural, domestic and aquatic animals. The probiotic may further contain one or more suitable excipients or flavoring agents suitable for oral administration to humans or animals.

The composition according to the invention can be used for the preparation of pharmaceutical compositions. Thus, the present invention also relates to a pharmaceutical composition containing a composition according to the invention and a pharmaceutically acceptable excipient or carrier.

Compositions containing the compounds of the invention can be prepared in various forms, for example, in the form of a tablet, capsule or powder. Examples of excipients that may be present in such compositions are diluents (eg, starch, cellulose derivatives or sugar derivatives), stabilizer (eg, hygroscopic fillers such as silica or maltodextrin), lubricant (eg, magnesium stearate), buffer (eg , phosphate buffer), binder, coating material, preservative or suspending agent. Suitable fillers are well known to those skilled in the art.

More specifically, the present invention relates to the composition described above, wherein said composition contains a total of 10 ⁵ -10 ¹¹ colony forming units of bacteria according to the invention.

In this document and in the claims, the verb “to include” and its grammatical forms are used in their non-limiting sense to mean that the elements following the verb are included, but not those not specifically stated are excluded. Additionally, the verb “consist of” may be replaced by “consist substantially of,” which means that the composition as defined herein, in addition to the components specifically stated, may contain additional component(s) that do not change (have) the unique properties of the invention. In addition, if a reference is made to an element preceded by the indefinite article "a" or "an", this does not exclude the possibility that more than one element is present, unless the context of the description specifically states that one and only one must be present. one element. Thus, the indefinite article "a" or "an" usually means "at least one."

All patents and literature references cited herein are hereby incorporated by reference in their entirety. The following examples are presented for illustrative purposes only and should in no way be construed as limiting the scope of the present invention.

Examples

Example 1. Preparation of a composition according to the invention

1.1. Isolation of bacteria for composition

A young, healthy donor who had not previously received antibiotic therapy was selected to inoculate the SHIME® model. By regulating several operating parameters of the SHIME® model (Figure 1, Van den Abbeele et al., 2010), it is possible to enrich and select gut microbiota networks that have beneficial effects on human health, such as microbiotas involved in dietary fiber fermentation, metabolism bile acids, in the breakdown of lactose, etc. The SHIME® system has been used to isolate bacterial strains with different functional properties, such as fiber-degrading strains (e.g. Bifidobacteria, Bacteroides ), fermenting strains (e.g. Escherichia coli ) or lactate-producing strains (e.g. Lactobacilli, Pediococci and Enterococci ). butyrate (e.g. Anaerostipes caccae, Butyricicoccus pullicaecorum, Faecalibacteruim prausnitzii, Roseburia hominis, Roseburia inulinivorans, Clostridium butyricum ) and propionate producers (e.g. Bacteroides thetaiotaomicron, Bacteroides vulgatus, Roseburia inulinivorans, Akkermansia muciniphila ). Selective media can be selected for these purposes, such as LAMVAB ( Lactobacilli ; Hartemink et al., 1997), RB ( Bifidobacteria ; Hartemink et al. 1996), enterococci media ( Enterococci ; Possemiers et al., 2004), TBX { Escherichia coli ; Le Bon et al., 2010), BBE ( Bacteroides fragilis group; Livingston et al., 1978), minimal mucin medium ( Akkermansia ; Derrien et al., 2004), M2GSC (butyrate producers; Barcenilla et al. 2000) or lactate-containing SHIME minimal medium (butyrate producers), SHIME minimal medium containing succinate and fucose (propionate producers), sulfate-rich minimal medium (sulfate reducers), SHIME minimal medium containing arabinoxylan and blood agar plates (Prevotella). In addition to SHIME, bacteria were also isolated directly from a fresh fecal sample from a healthy donor using the same strategy.

In practice, 10-fold dilutions of samples collected from SHIME colonic compartments or homogenized fecal samples were obtained and these samples were spread on agar plates containing the specific media described above. The plates were incubated at 37°C under appropriate conditions for cultivating bacteria of various groups. After incubation, approximately 30 colonies per bacterial group were collected, and these colonies were incubated in the appropriate liquid culture medium under appropriate conditions. SCFA concentrations in overnight cultures were analyzed by gas chromatography as described by Possemiers et al. (2004). In addition, a sample of liquid cultures was used for phylogenetic analysis. DNA was extracted as described by Possemiers et al., 2004, and nearly all 16S rRNA sequences were amplified for each isolate using the universal eubacterial primers fD1 and rD1 (Weisburg et al., 1991). After purification, DNA samples were submitted for sequencing. The obtained sequences were aligned with the available sequences to identify each isolate using the BLAST software package ( http://blast.ncbi.nlm.nih.gov/Blast.cqi ).

1.2. Preparation of a composition according to the invention

Pure cultures isolated from the SHIME® reactor and feces (as described in Example 1.1) were used to combine different bacterial strains into actual functional microbial networks. In addition, pure cultures were obtained from culture collections such as BCCM/LMG ( http://bccm.belspo.be ) and DSMZ ( www.dsmz.de ).

Short chain fatty acids (SCFAs) are the end products of dietary fiber fermentation in gut microbiota and are known to have several beneficial effects on host health. The main SCFAs produced are acetate, butyrate and propionate in an approximate molar ratio of 60:20:20. Acetate can be absorbed by the intestine and is used by the host as an energy substrate, and butyrate is a major source of energy for the intestinal epithelium and has been shown to have protective effects against inflammation and colon cancer. Propionate has the same local activity in the intestine as butyrate, but it is also transported to the liver, where it has been shown to have positive cholesterol-lowering effects and regulate blood glucose levels.

Given the important and diverse physiological roles of SCFA, disruption of this intestinal microbial function (eg, in gastrointestinal disorders) can have a significant impact on the health of the host. Therefore, in this example, screening was performed to obtain a composition that can induce the highest level of total SCFA production and in the most preferred ratios. In the latter case, butyrate is considered to be the most interesting of all the various SCFAs produced. In addition, the effects of different formulations on the integrity of the intestinal barrier were assessed by co-culturing epithelial and immune cells.

In practice, a total of 20 isolates (referred to as “isolates-X”) with the most interesting fermentation profiles were obtained from glycerol stock solutions, after a round of isolation and selection as described in 1.1, or they were ordered from culture collections, after which these isolates cultured under appropriate optimal culture conditions to obtain homogeneous suspensions of bacterial strains.

No. View Strain No. View Strain 1 Lactobacillus plantarum Isolate-1 eleven Faecalibacterium prausnitzii Isolate-11 2 Clostridium bolteae Isolate-2 12 Roseburia inulinivorans Isolate-12 3 Desulfovibrio desulfuricans Isolate-3 13 Ruminococcus spp. Isolate-13 4 Akkermansia muciniphila Isolate-4 14 Lacobacillus acidophilus Isolate-14 5 Coprococcus spp. Isolate-5 15 Enterococcus faecium Isolate-15 6 Roseburia hominis Isolate-6 16 Butyrivibrio fibrisolvens Isolate-16 7 Bacteroides thetaiotaomicron Izolyat-7 17 Eubacterium limosum DSM20543 Nissle 8 Clostridium butyricum Isolate-8 18 Escherichia coli 1917 9 Anaerostipes caccae Isolate-9 19 Eubacterium rectale DSM17629 10 Bifidobacterium adolescentis Isolate-10 20 Butyricicoccus pullicaecorum Isolate-17

Isolates were pooled in numbers ranging from 2 to 10 in a series of 98 separate initial screening experiments. For each experiment, fermentation was started in sterile incubation bottles containing sterilized SHIME® culture medium adjusted to pH 6.8 with KH ₂ PO ₄ /K ₂ HPO ₄ and purged with nitrogen. The sterilized medium was then inoculated with a 10% (v/v) mixed inoculum consisting of equal volumes of the selected species. Incubation bottles were purged with nitrogen to ensure anaerobic conditions and incubated at 37°C (90 rpm). Samples were analyzed after 24 hours for SCFA production. Compositions with the highest levels of butyrate production were then selected and used in a final experiment with 23 different batches of bacteria (referred to as MX-Y, where X=number of isolates present in the composition and Y=unique composition A, B, C, etc. .p. with isolates X).

These 23 combinations were again incubated as described above. After 24 hours, samples were taken for SCFA analysis and combination with the Caco-2 and THP1 cell coculture model as described by Possemiers et al. (2013). The endpoint of the latter experiment is the measurement of trans-epithelial electrical resistance (TEER) as a measure to assess protective effects on intestinal barrier function.

In fig. Figure 2 shows the butyrate levels obtained after 24 hours of incubation for 23 different formulations and their effect on TEER values. A strong change was observed in butyrate levels and the effect on intestinal barrier function, with combinations with the highest butyrate levels not necessarily inducing the highest protective effect on TEER levels as shown by the various evaluation ratings. Surprisingly, one composition of 7 different isolates (called M7-B in Figure 2) ranked first for butyrate levels at 24 hours, and especially for protective effects on intestinal barrier function. This composition contained 6 SHIME isolates and one culture obtained from a human fecal sample. Sequencing of the 16S rRNA gene and comparison of its sequence with the NCBI BLAST database sequence revealed that M7-B consists of new SHIME isolates of Lactobacillus plantarum, Faecalibacterium prausnitzii, Roseburia inulinivorans, Roseburia hominis, Akkermansia muciniphila and Anaerostipes caccae and a new fecal isolate of Butyricicoccus pullicaecorum . It is interesting to note that all of the new isolates were present in at least one of the other compositions shown in FIG. 2, but none of the other compositions showed the same effectiveness on butyrate production and protective effect on TEER values. This indicates that the observed effect is not due to any of the specific species present in the composition, and only a specific combination of these 7 bacteria unexpectedly produced positive results.

7 new isolates were deposited in the bacterial collection BCCM/LMG (Ghent, Belgium) with registration numbers: Faecalibacterium prausnitzii LMG P-29362, Butyricicoccus pullicaecorum LMG P-29360, Roseburia inulinivorans LMG P-29365, Roseburia hominis LMG P-29364, Akkermansia muciniphila LMG P-29361, Lactobacillus plantarum LMG P-29366 and Anaerostipes caccae LMG P-29359.

Since an additional experiment revealed an unexpected synergistic effect between the 7 isolates and the need for the presence of each of the species, an experiment was conducted in which each time one of these isolates was removed from the original composition consisting of 7 isolates (that is, it was eliminated) . In practice, fermentation was started again in sterile incubation bottles containing sterilized SHIME® culture medium adjusted to pH 6.8 with KH ₂ PO ₄ /K ₂ HPO ₄ and purged with nitrogen. The sterilized medium was then inoculated with a 10% (v/v) mixed inoculum consisting of equal volumes of 6 of the 7 isolates. The complete composition of 7 isolates was used as a control and a total of 8 parallel incubations were performed. Incubation bottles were purged with nitrogen to ensure anaerobic conditions and incubated at 37°C (90 rpm). Samples were analyzed after 24 hours and 48 hours for butyrate production. As shown in FIG. 3, removal of only one species from the original composition resulted in a significant reduction in butyrate production levels after 24 hours for all compositions of the 6 species to 80% of the butyrate production level in the original composition. Additionally, after incubation for 48 hours, butyrate levels were significantly lower for all 6-species compositions, with the exception of the Roseburia hominis- free composition. This confirms that all isolates of the composition are essential to achieving the full potential of the composition. Since only one composition, not containing Roseburia hominis , had functional properties similar to those of the full composition after 48 hours of incubation, it can also be predicted that the second most effective composition is a 6-species composition consisting mainly of Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburia inulinivorans, Akkermansia muciniphila, Lactobacillus plantarum and Anaerostipes caccae .

1.3. Preparation of a composition according to the invention

A composition consisting of Lactobacillus Plantarum, Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburia inulinivorans, Roseburia hominis, Akkermansia muciniphila and Anaerostipes caccae species was prepared using 3 different strategies. These strategies include: 1) culturing individual composition species and then mixing them, 2) co-culturing the composition species in a multi-stage fermenter (ie, the SHIME® in vitro model as described above), and 3) co-cultivating the composition species in a single-stage fermenter.

In the first strategy ( the “microcommunity” strategy ), selected species were isolated from glycerol stock solutions and cultured under appropriate optimal culture conditions to obtain homogeneous suspensions of bacterial strains. To assess their functional activity, a mixed inoculum was obtained, consisting of equal volumes of the selected species. This inoculum at 10% (v/v) was added to sterile incubation bottles containing sterilized SHIME® culture medium adjusted to pH 6.8 with KH ₂ PO ₄ /K ₂ HPO ₄ . Incubation bottles were purged with nitrogen to ensure anaerobic conditions and incubated at 37°C (90 rpm). At specified 16-hour intervals, 40% (v:v) of the culture medium was replaced with SHIME® conditioned growth medium. SHIME® conditioned culture medium was prepared by incubating 700 ml of normal SHIME® food (pH 2) for 1 hour at 37°C, followed by the addition of 300 ml of pancreatic juice (pH 6.8), to which 25 g/l was added NaHCO ₃ , 23.6 g/l KH ₂ PO ₄ and 4.7 g/l K ₂ HPO ₄ . Samples were analyzed over 5 days for SCFA production (Fig. 4). Butyrate levels reached 7 mM after 24-hour incubation of microcommunities, and a maximum of 14 mM after 5 days.

In the second strategy (i.e., the “collaborative” strategy or the strategy “where the specified bacteria were cultured together in a dynamic gastrointestinal simulator prior to administration”), selected species were isolated from glycerol stock solutions and cultured under appropriate optimal culture conditions to obtain homogeneous suspensions of bacterial strains. The strains were then mixed and plated in triplicate in the SHIME® system (Van den Abbeele et al., 2010) consisting of one area of the colon at pH 6.15–6.4. To obtain a functional collaboromic composition, the adaptation period was two weeks. The need and relevance of such an adaptation period was clearly demonstrated by the change in SCFA profiles during the cultivation of the selected species composition (Fig. 5). Initially, these compositions require a certain adaptation time to adapt to each other and become active in converting the supplied substrates into SCFA. However, 4-6 days after inoculation, SCFA production by this composition began to stabilize, resulting in high levels of butyrate being detected. On the last day of incubation (day 14), each of the three incubations resulted in highly similar stable and highly active functional compositions with butyrate levels of 19 mM.

After freezing the stabilized collaborome at -80°C as a glycerol stock solution and then thawing it for use as an inoculum in the same manner as for the microcommunity strategy, it was unexpectedly found that butyrate levels increased more rapidly and reached 25% at the same incubation conditions, as in the case of using the strategy of microcommunities of individual species (Fig. 4). Butyrate levels reached 12 mM after only 24 hours of microcommunity incubation and reached a maximum of 19 mM after 2 days.

In a third strategy, production of the composition was accomplished using an optimized one-stage fermenter operating in a fed-batch mode (ie, an alternative "collaboration" strategy or a strategy "where the bacteria were cultured together in the fermenter prior to injection"). Selected species were isolated from glycerol stock solutions and cultured under appropriate optimal culture conditions to obtain homogeneous suspensions of bacterial strains. Fermentation was started in sterile incubation bottles containing sterilized SHIME® culture medium adjusted to pH 6.8 with KH ₂ PO ₄ /K ₂ HPO ₄ and purged with nitrogen. The sterilized medium was then inoculated with a 10% (v/v) mixed inoculum consisting of equal volumes of the selected species. Incubation bottles were purged with nitrogen to ensure anaerobic conditions and incubated at 37°C (90 rpm). At specified 16-hour intervals, 40% (v:v) of the culture medium was replaced with SHIME® conditioned growth medium. SHIME® conditioned culture medium was prepared by incubating 700 ml of normal SHIME® food (pH 2) for 1 hour at 37°C, followed by the addition of 300 ml of pancreatic juice (pH 6.8), to which 25 g/l was added NaHCO ₃ , 23.6 g/l KH ₂ PO ₄ and 4.7 g/l K ₂ HPO ₄ .

As shown in FIG. 6, the production of total SCFAs and the ratios of SCFAs produced by this composition stabilized after 6 replacement cycles. When reinoculated using the same strategy described above, the stabilized collaborator produced maximum SCFA production (acetate/propionate/butyrate ratio was approximately 12/14/74) 2 days earlier compared to the same series of species in the microcommunity strategy, and butyrate production was 25% higher.

Example 2: Experiments in vitro

2.1 Effect of adding a functional composition to complex gut microbial communities

This experiment demonstrated that the functional composition is active when inoculated into a mixed intestinal microbial community where there is strong competition for binding colonic substrates with members of this complex intestinal community, which is estimated to consist of 500-1000 species of microorganisms. To address this issue, an experiment was conducted in small incubation bottles using a composition containing Lactobacillus Plantarum, Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburia inulinivorans, Roseburia hominis, Akkermansia muciniphila and Anaerostipes caccae , where the said composition was prepared using a collaboration strategy as described in Example 1.3. The pre-adapted composition in increasing concentrations (0, 4 and 20%) was washed in PBS and added to three different media such as:

1) Sterile basal medium [2 g/L peptone, 2 g/L yeast extract, 2 ml/L Tween 80, 10 µL/L vitamin K1, 500 mg/L L-cysteine•HCl, 100 mg/L NaCl, 40 mg/l K ₂ HPO ₄ , 40 mg/l KN ₂ PO ₄ , 10 mg/l MgSO ₄ •7H ₂ O, 6.7 mg/l CACl ₂ •2H ₂ O, 1.5 mg/l resazurin, 50 mg/l hemin (50 mg/l) - pH 5.5]+6 g/l starch;

2) Basal medium + 20% fecal suspension (prepared as described by De Boever et al., 2000).

3) Basal medium + 20% SHIME® colon suspension containing complete microbiota.

Increasing the concentration of the preadapted butyrate-producing community from 0% to 4% to 20% resulted in a proportional increase in absolute butyrate levels (Fig. 7). This was observed not only in a sterile environment, but also in an environment supplemented with mixed microbiota obtained from a fecal sample or SHIME® colon area. Thus, this experiment showed that this composition is not only active when it is present in the non-competitive environment of the colon, but it also produces higher levels of butyrate when introduced into a mixed microbiota, where multiple gut microbes compete to bind to the same the same nutrients. Additionally, not only did butyrate levels increase, but propionate production also increased greatly. This cumulative increase in these levels and decrease in acetate levels upon incubation suggests that this composition may modulate overall microbial fermentation profiles, resulting in more health-promoting profiles.

2.2. Efficiency of a functional composition in restoring the metabolic functions of the intestinal microbial community subjected to antibiotic-induced dysbiosis

Antibiotic use has been suggested to cause severe disruption of the intestinal microbial community. It has been shown that a microbial composition subjected to dysbiosis is more susceptible to infection by pathogens. Additionally, a number of gastrointestinal diseases, such as inflammatory bowel disease, are correlated with dysbiotic microbial compositions, highlighting the importance of the gut microbiome for human health. Recovery of taxonomic composition, and especially functionality, after long-term antibiotic use usually takes three months, and reaches the state observed before treatment, namely the gut microbial community of a healthy individual (Panda et al., 2014). Decreasing recovery time from antibiotic exposure may thus reduce the risk of developing serious infections and improve the overall health of the host. Accordingly, the observed functional activity of the selected composition can be considered as a promising strategy for increasing the level of recovery of microbial communities after antibiotic-induced dysbiosis and reducing the risk of infection.

In this example, antibiotic-induced dysbiosis was simulated in the SHIME® system in vitro by dosing the appropriate antibiotics. The purpose of this experiment was to evaluate the restoration of typical “healthy” metabolite profiles in a simulated colonic environment following administration of a functional formulation. In addition, the purpose of this experiment is to differentiate the effectiveness of a composition obtained using either a “microcommunity” strategy or a “collaborative” strategy (see Example 1.3). This experiment was repeated using a composition containing Lactobacillus plantarum, Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburia inulinivorans, Roseburia hominis, Akkermansia muciniphila and Anaerostipes caccae . To more accurately mimic the full functional profile of the gut microbiome, the composition used in this particular experiment was additionally spiked with Escherichia coli, Enterococcus faecium, Lactobacillus mucosae, Bifidobacterium adolescentis, Bifidobacterium longum, Bacteroides thetaiotaomicron , and Bacteroides vulgatus .

In practice, SHIME® vessels (pH 6.15-6.40) were inoculated with fecal material and left to stabilize for 14 days (in the M-SHIME® system - Van den Abbeele et al., 2012). After a 2-week control period, SHIME® colon microbiota was treated with an antibiotic cocktail (40/40/10 mg/L amoxicillin/ciprofloxacin/tetracycline, respectively) to induce dysbiosis. The next day, the dysbiotic microbiota was treated for 5 days with a functional composition obtained using either a “microcommunity” strategy or a “collaborome” strategy. The endpoint of the study is to evaluate the restoration of typical “healthy” SCFA metabolite profiles in a simulated colonic environment. The SHIME® control vessel was used to simulate the spontaneous recovery of metabolic activity of the gut microbial community after antibiotic exposure without administration of the formulation. Results are expressed as delta SCFA levels in SHIME at each time point compared to results obtained before antibiotic administration (Fig. 8).

After treatment of SHIME® vessels with antibiotics, a significant reduction in the production of acetate, propionate and butyrate was observed. This observation confirms the disruption of the gut microbial community. Restoration of the metabolite profile (under SCFA production conditions) to the pre-treatment state is illustrated in FIG. 8 as changes in acetate, propionate and butyrate levels over 5 days. This indicates that the restoration of functionality was slow in the case of the control (without administration of the composition), and at the same time, complete restoration of acetate and propionate levels was not observed within 5 days. It is interesting to note that treatment with the composition resulted in faster recovery compared to control for all 3 SCFAs. Additionally, the formulation using the microcommunity strategy induced complete recovery of propionate and butyrate levels after 5 days and 3 days, respectively, and the formulation using the collaborome strategy induced a faster recovery compared to the recovery from administration. "microcommunity" strategy, and in this case, full restoration of propionate and butyrate levels was achieved after 4 days and 2.5 days, respectively. Finally, the “collaborome” strategy also resulted in increased final activity with increased levels of propionate and butyrate, in contrast to the “microcommunity” strategy. These results support the possibility of using this composition to restore antibiotic-mediated microbial dysbiosis. Moreover, these results clearly showed that pre-adaptation using the “collaborome” strategy achieves more efficient recovery of microbial SCFA production after antibiotic exposure compared to the “microcommunity” strategy.

2.3: Efficiency of a functional composition in restoring the metabolic functions of the intestinal microbial community subjected to dysbiosis in inflammatory bowel diseases

Inflammatory bowel disease (IBD) is associated with disruption of host microbial interactions, which in turn is, at least in part, associated with dysbiosis of the gut microbiota. In the latter case, for example, there is a decrease in the amount of butyryl-CoA: acetate-CoA transferase and propionate kinase (Vermeiren et al., FEMS 2011), which in turn negatively affects the ability to produce balanced SCFA. Given the important influence of SCFA on the normal development and maintenance of the intestinal microbiota, it can be concluded that restoring the composition and functionality of the microbiota, namely the production of SCFA, may have a positive effect on the symptoms associated with IBD. Accordingly, the observed functional activity of the selected composition may be a promising strategy to accelerate the restoration of microbial communities during dysbiosis in the case of IBD, used as a main strategy to restore and maintain a healthy intestinal barrier.

In this example, IBD-associated dysbiosis was modeled using the M-SHIME® in vitro model as previously described (Vigsnaes et al. 2013). The purpose of this experiment is to evaluate the recovery of microbiota, namely SCFA profiles, in a simulated colonic environment following administration of a functional formulation. In addition, the purpose of this experiment is to differentiate the effectiveness of a composition obtained using either a “microcommunity” strategy or a “collaborative” strategy (see Example 1.3). This experiment was repeated using a composition containing Lactobacillus plantarum, Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburia inulinivorans, Roseburia hominis, Akkermansia muciniphila and Anaerostipes caccae .

In practice, SHIME® vessels (pH 6.15–6.40) were inoculated with fecal material collected from a patient with ulcerative colitis (M-SHIME® system - Van den Abbeele et al., 2012). Simultaneously, to simulate administration, a single dose of the functional formulation prepared using either the microcommunity or collaboration strategy was added to the colon area. The third experiment was carried out in parallel as a control experiment without the introduction of the composition. The production of acetate, propionate and butyrate was observed 1 and 2 days after administration of the composition.

The results obtained are presented in Fig. 9: Administration of the composition obtained using the microcommunity strategy resulted in an increase in the production of SCFA (mainly acetate and butyrate) on day 1, but this effect was no longer observed on day 2. This indicates that this composition is functionally active in the microbiome environment in IBD. It is interesting to note that the effect on propionate and butyrate production was much more pronounced after administration of the composition obtained using the "collaborome" strategy, that is, in this case, there was a 4-fold and 3-fold increase in the levels of propionate and butyrate, respectively, compared to with IBD control. In contrast to the composition obtained using the microcommunity strategy, this effect was still quite pronounced at day 2 and corresponded to lower acetate production (as indicated by an increase in the level of cross-feeder culture and thus an increase in the network of microorganisms). These results confirm the possibility of using this composition to eliminate IBD-associated microbial dysbiosis. Moreover, these data clearly indicate that pre-adaptation using the “collaborome” strategy resulted in more effective restoration of microbial SCFA production in IBD compared to the “microcommunity” strategy.

2.4. The effectiveness of the functional composition in inhibiting the growth of vegetative Clostridium difficile in modeling analysis in vitro

In this example, a Clostridium difficile challenge test was performed to determine whether the functional composition is not only functionally active in intestinal conditions, but whether it can also protect the intestinal environment from infections. In such a challenge test, the composition was infected with vegetative cells of Clostridium difficile ( Cdif ) to evaluate their ability to inhibit the growth of Cdif under simulated gastrointestinal conditions. In addition, an experiment was conducted to differentiate the effectiveness of a composition obtained using either a “microcommunity” strategy or using a “collaboromy” strategy (see example 1.3). This experiment was repeated using a composition containing Lactobacillus plantarum, Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburia inulinivorans, Roseburia hominis, Akkermansia muciniphila and Anaerostipes caccae .

In practice, a glycerol stock solution of Clostridium difficile (LMG 21717 ^T ) was thawed and inoculated into a bottle containing broth enriched with clostridium medium (RCM), which was then purged with nitrogen to create anaerobic conditions. The bottle was incubated in a shaker incubator (90 rpm) for 24 hours, and 10% of the grown culture was reinoculated into RCM broth. 24 hours after cultivation, the homogenized C. difficile culture was aliquoted (in triplicate) into bottles (10% v/v) containing:

1) basal medium (control);

2) a basal medium containing a composition obtained using a “microcommunity” strategy;

3) basal medium containing a composition obtained using the “collaborome” strategy;

4) basal medium containing SHIME® colon suspension.

The bottles were incubated at 37°C in a shaking incubator (90 rpm). At regular intervals, the sample was collected and immediately frozen at -80°C until C. difficile was quantified using qPCR analysis based on detection and quantification of the triose phosphate isomerase gene. For this purpose, genomic DNA was extracted as described by Boon et al. (2003). The amplification reaction was carried out using forward and reverse oligonucleotides: 5'-TATGGACTATGTTGTAATAGGAC-3' (forward) and 5'-CATAATATTGGGTCTATTCCCTAC-3' (reverse). The absolute amount of PCR product was calculated by constructing a standard curve.

In this controlled in vitro simulation assay, growth of C. difficile was observed in basal medium after 48 hours of incubation, confirming the presence of a control in the in vitro simulation assay. SHIME® colon suspension (as a real fecal transplant model) exhibited the greatest inhibition of C. difficile growth after 48 hours of incubation (ie, 58%). It is interesting to note that a similar result was obtained for the composition obtained using the "collaborome" strategy, as indicated by approximately 53% inhibition of C. difficile growth. The lowest effect was observed when adding the composition obtained using the microcommunity strategy (ie, 23% growth inhibition). This experiment clearly demonstrated that the growth of C. difficile was significantly inhibited by the composition, and that such inhibition was most pronounced when the composition was pre-adapted using a “collaborome” strategy.

2.5. Effect of a functional composition on a biomarker of intestinal barrier functioning and on intestinal immunity in the host

Examples 2.1-2.3 show that the composition is functionally active in complex intestinal conditions and can restore intestinal metabolite profiles, with the highest activity observed when produced using the “collaborome” strategy. This may, in turn, have a beneficial effect on the intestinal epithelium and thus on the functioning of the intestinal barrier and local immunity.

In this example, a combination of samples taken from previous experiments in an established coculture model of enterocytes (Caco-2 cells) and macrophages (THP1) was used to evaluate this possibility (Possemiers et al. 2013). In this model, stimulation of THP1 cells by lipopolysaccharides leads to increased levels of proinflammatory cytokine production, which in turn will disrupt the enterocyte layer, creating a so-called leaky gut condition. The effect on leaky gut is measured by assessing the effect of transepithelial electrical resistance (TEER) [a measure of the effectiveness of the intestinal barrier] and the level of inflammatory cytokine production compared to control.

In practice, samples collected 1 day after the M-SHIME experiment, as described in Example 2.3, were combined with the leaky gut co-culture model.

2.6. The influence of variations in strain identity on the functional activity of the composition

To determine whether the unexpected synergistic effect of the 7 isolates in the composition was strain-specific or could also be achieved using other strains of the same species, an additional experiment was conducted. In this example, two different compositions were prepared using a "collaboration" strategy (see Example 1.3). Composition 1 contains the specific isolates described in example 1.2, and composition 2 consists of strains of the same species obtained from the culture collection:

- Composition 1: Faecalibacterium prausnitzii LMG P-29362, Butyricicoccus pullicaecorum LMG P-29360, Roseburia inulinivorans LMG P-29365, Roseburia hominis LMG P-29364, Akkermansia muciniphila LMG P-29361, Lactobacillus plantarum LMG P-29366 and Anaer ostipes caccae LMG P -29359;

- Composition 2: Lactobacillus plantarum ZJ316, Faecalibacterium prausnitzii (DSMZ 17677), Butyricicoccus pullicaecorum (LMG 24109), Roseburia inulinivorans (DSMZ 16841), Roseburia hominis (DSMZ 16839), Akkermansia muciniphila (DSMZ 22959) and Ana erostipes caccae (DSMZ 14662).

In practice, selected species were isolated from glycerol stock solutions and cultured under appropriate optimal growth conditions to obtain homogeneous suspensions of bacterial strains. The strains were then mixed to form Composition 1 and Composition 2, respectively, and each was seeded in triplicate in the SHIME® system (Van den Abbeele et al., 2010), consisting of one area of the colon at pH 6.15-6. 4. Butyrate production profiles were observed for 14 days.

It is interesting to note that the dynamics of butyrate production were very similar for both formulations, that is, there were large fluctuations at first and then, after approximately 6 days, butyrate levels stabilized. At the end of the experiment (day 14), butyrate levels for composition 1 were 19.3 mM and levels for composition 2 were 18.8 mM. This indicates that the synergistic effect observed in the composition of Example 1.2 can be reproduced using different strains obtained from the same species.

Example 3. Experiments in vivo

3.1. Mouse model of antibiotic-induced destruction of the gastrointestinal microbiota

In this example, an experiment was conducted to determine whether the functional composition could restore the metabolic capacity of the gut microbiome after antibiotic-induced dysbiosis, also in in vivo conditions.

This example used a composition containing Lactobacillus plantarum, Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburia inulinivorans, Roseburia hominis, Akkermansia muciniphila and Anaerostipes caccae and obtained using the "collaborome" strategy of Example 1.3. Additionally, to determine whether a more accurate mimic of the full functional profile of the gut microbiome was needed, an additional experiment was conducted using a formulation supplemented with Escherichia coli, Enterococcus faecium, Lactobacillus mucosae, Bifidobacterium adolescentis, Bifidobacterium longum, Bacteroides thetaiotaomicron and Bacteroides vulgatus (this composition is called a composition with “extended composition”).

In practice, the "formulation" and "formulation-extended" were prepared fresh using the "collaborome" strategy, washed twice in PBS (in an anaerobic chamber to ensure anaerobic conditions), concentrated in 100 μl and administered to mice via gavage, as soon as possible. Mice (C57/ND6) at least 5 weeks old were purchased and these mice were maintained in pathogen-free conditions and fed standard chow. Experiments on mice were performed in accordance with protocols approved by the Animal Test Ethics Committee of Ghent University, Belgium. To induce dysbiosis caused by antibiotics, a dose of the antibiotic clindamycin was introduced into drinking water at a concentration of 250 mg/l. 5 days after administration of the antibiotic, the stomach contents of the mice were neutralized with NaHCO ₃ , after which the mice (10 mice per group) were administered orally through a gastric tube for 5 consecutive days:

1) composition in physiological solution;

2) a composition with an “expanded composition” in saline solution and

3) saline solution (control).

The normal group (which did not receive antibiotics but was treated with saline) was included as a control to exclude variability occurring after gavage insertion. Fecal samples (approximately 100 mg/mouse) were collected during the experiment and stored at -80°C for subsequent analyses.

SCFA profiles obtained from fecal samples collected from mice of the same groups showed that 5 days of antibiotic treatment resulted in a significant reduction in the levels of butyrate and propionate production to the extent that only acetate was produced (Fig. 10). As shown in FIG. 10, spontaneous recovery of metabolic functions occurred slowly and did not begin until approximately 5 days (D10) after the last antibiotic treatment, although the molar ratios of the three major SCFAs (acetate, propionate, and butyrate) did not reach the state observed before antibiotic administration. However, after treating mice with the “formulation” or “expanded composition” obtained using the “collaborome” strategy, restoration of butyrate metabolism began as early as approximately 3 days (d8) after antibiotic treatment. Moreover, metabolic activity in mice treated with both formulations was almost completely restored 5 days after the last dose of antibiotics (d10) with good production of both propionate and butyrate. The expanded composition contained a higher diversity of acetate and propionate producers compared to the conventional composition, which was also evidenced by a slightly different fermentation profile on day 10 of the experiment. In conclusion, this example provides in vivo evidence that the functional formulation is effective in achieving faster and more potent restoration of intestinal metabolite profiles following antibiotic-induced dysbiosis. In addition, changing the exact combinations of species in the composition allows you to adjust the final result with respect to specific metabolite profiles.

3.2. Mouse TNBS model of inflammation

The TNBS ( 2,4,6-trinitrobenzenesulfonic acid ) model is a widely used colitis model that mimics several features of Crohn's disease (Scheiffele et al., 2001), including weight loss, bloody diarrhea, and thickening of the intestinal wall. Histopathological analysis of TNBS revealed foci of transmural inflammation of the intestine with the formation of deep ulcers, the classic features of which are observed in patients with CD. This makes the TNBS model a good candidate for in vivo assessment of the ability of a functional composition to prevent and/or reverse damage to the intestinal mucosa in IBD and promote the maintenance/development of a healthy intestinal barrier.

In this example, a composition containing Lactobacillus plantarum, Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburia inulinivorans, Roseburia hominis, Akkermansia muciniphila and Anaerostipes caccae was used to evaluate the beneficial effects after applying the TNBS model. In addition, an experiment was conducted to differentiate the effectiveness of a composition obtained either using the “microcommunity” strategy or using the “collaboromy” strategy (see example 1.3). Colitis in animals was caused by rectal instillation of TNBS, that is, mucosal sensitizing agents diluted in ethanol. The introduction of ethanol is a necessary condition for the destruction of the colonic mucosal barrier to ensure the penetration of TNBS into the lamina propria. TNBS haptenizes localized colon and intestinal microbial proteins, causing them to become immunogenic and trigger natural and adaptive immune responses in the host.

In practice, 8- to 10-week-old male C57BL6/J mice were housed in a temperature-controlled chamber at 20°C on a 12-hour day-night cycle. Animals had free access to water and commercially available food. Mice were randomly assigned to cages to avoid confinement effects. The experiment began after acclimatization for 1 week. Each group (n=9/group) was treated for 5 consecutive days using an oral gavage. Prophylactic dosing for all treatments began 1 day before rectal administration of 2 mg TNBS/50%EtOH and continued for 4 days after TNBS administration, and then mice were sacrificed. The following processing was carried out:

1) TNBS+ composition obtained using the “microcommunity” strategy in saline solution;

2) TNBS+ composition obtained using the “collaborome” strategy in saline solution; And

3) TNBS+saline (control).

The normal group (which was not treated with TNBS but was treated with saline) was included as a control to exclude variability occurring after probe insertion. The endpoint of the study was daily monitoring of disease activity (before daily treatment) by measuring body weight, presence of blood in feces (ColoScreen) and assessment of general appearance.

The results of this example are shown in FIG. 11. The control group treated with vehicle (saline) without TNBS showed no weight loss or disease activity, and the control group treated with TNBS immediately experienced an 8% weight loss on day 1 and a strong increase in disease activity. Both weight loss and disease activity were partially restored at the end of the study. It is interesting to note that the compositions provided a strong protective effect against weight loss and disease activity, yet the extent of this protective effect depended on the formulation strategy. An initial weak protective effect was observed on day 1 for the microcommunity strategy, as indicated by reduced weight loss and disease activity, but this protective effect was no longer observed on subsequent days of the study. In contrast, administration of the composition obtained using the "colaborome" strategy had a strong prophylactic effect, preventing weight loss and disease activity on day 1, compared with the TNBS control, and a more rapid and complete recovery was observed at the end of the study, which indicated a return of disease activity to the level observed with vehicle control. In conclusion, this example provides in vivo confirmation that the functional composition is effective in achieving more rapid prevention and more rapid and effective resolution of intestinal inflammation and disease activity in TNBS-induced colitis. Moreover, these results clearly demonstrated that pre-adaptation using the collaborative strategy is more effective than adaptation using the micro-community strategy.

3.3. Mouse DSS model of inflammation

The DSS model of chronic colitis is a widely used model of colitis that mimics some of the features of Crohn's disease, including weight loss and bloody diarrhea. According to histopathological analysis, long-term administration of DSS caused intestinal inflammation with typical structural changes such as crypt curvature, (sub)mucosal inflammatory cell infiltration and fibrosis, i.e., features observed in CD patients. This makes the DSS model a good candidate for in vivo assessment of the ability of a functional composition to prevent and/or reverse damage to the intestinal mucosa in IBD and promote the maintenance/development of a healthy intestinal barrier.

In this example, a composition containing Lactobacillus plantarum, Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburia inulinivorans, Roseburia hominis, Akkermansia muciniphila and Anaerostipes caccae was used and obtained using a “collaborome” strategy (see Example 1.3) to evaluate the beneficial effects after application of DSS - models of chronic colitis. Colitis was induced in animals by repeated administration of DSS to drinking water (0.25% challenge). This experiment was carried out for a total of 8 days with 3 cycles of DSS administration and recovery.

In practice, 6-week-old male C57BL6/J mice were housed in a temperature-controlled chamber at 20°C on a 12-hour day-night cycle. Animals had free access to water and commercially available food. Mice were randomly assigned to cages to avoid confinement effects. The experiment began after acclimatization for 1 week. Each group (n=10/group) was treated 3 times a week for 8 consecutive days using an oral gavage. The prophylactic dose for all treatments began 1 week before the first DSS cycle. The first DSS cycle began at week 2, and this cycle included 1 week of DSS administration (0.25% in drinking water) followed by 2 weeks of recovery. After the first cycle, an identical second DSS cycle was performed. The third DSS cycle consisted of DSS administration for one week followed by recovery for one week, after which the animals were sacrificed. The following processing was carried out:

1) Control without DSS

2) DSS+composition obtained using the “collaborome” strategy in saline solution (3 times a week); And

3) DSS+saline (DSS-control).

The endpoint of the study was daily monitoring of the disease activity index (DAI) during each DSS cycle three times a week (before daily treatment) by measuring body weight, presence of blood in feces (ColoScreen) and assessment of general appearance. As shown in FIG. 12, in the case of the control group treated with vehicle (saline) without DSS, no effect on DAI was observed, while the control group treated with DSS showed a significant increase in DAI in each administration cycle. It is interesting to note that this composition provided a strong protective effect (approximately 25% reduction in DAI in each cycle) against disease activity. This also indicates that the functional composition is effective in achieving a strong protective effect against intestinal inflammation and disease activity in DSS-induced colitis.

Example 3.5. Model of mucosal inflammation

Mucositis is a clinical term used to describe damage to the mucous membranes following cancer therapy. Mucosal inflammation occurs throughout the gastrointestinal (GI) tract (including the mouth), the genitourinary tract, and to a lesser extent on other mucosal surfaces. Its severity and duration vary depending on the dose and type of medicine used.

An important feature of mucosal inflammation is that it limits the dose of chemotherapy. The GI crypt epithelium is particularly susceptible to chemotherapy toxicity, and symptoms include nausea and vomiting, abdominal pain, bloating, and diarrhea due to direct effects of cytotoxic substances on the mucosa. A rat model of 5-fluorouracil (5-FU)-induced intestinal mucosal inflammation was developed by Keefe et al. to evaluate the effects of chemotherapy on the gastrointestinal tract and is currently one of the most widely used models for studying mucosal inflammation in rats. induced by chemotherapy (Keefe 2004).

In this example, the basis for the experiment was a composition containing Lactobacillus plantarum, Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburia inulinivorans, Roseburia hominis, Akkermansia muciniphila and Anaerostipes caccae and obtained using the “collaborome” strategy of Example 1.3. Mucosal inflammation was induced by administering a single intraperitoneal dose of 5-FU.

In practice, a total of 30 rats were randomly assigned to control and experimental groups according to a specific time point. All rats in the experimental groups received one intraperitoneal dose of 5-FU (150 mg 5-FU/kg body weight). Rats in control groups were treated with the vehicle vehicle (dimethyl sulfoxide). After administration of chemotherapy drugs, study endpoints such as mortality, diarrhea and general clinical status were assessed four times within 24 hours. Rats included in the subgroups were sacrificed by bleeding and mixing the cervical vertebrae 24, 48 and 72 hours after drug administration. The primary endpoints of interest are weight change, diarrhea, and global clinical status (frailty score). Secondary endpoints include histological analysis of intestinal samples and data from the analysis of stool and intestinal mucosal microbiota.

In order to evaluate the effect of the composition in terms of prevention or amelioration of the assessed symptoms, some rats were administered the composition by oral gavage for 8 consecutive days. The prophylactic dose was administered 5 days before the start of 5-FU administration and continued for 3 days after 5-FU administration or until the rats were sacrificed. Control animals were not administered the composition.

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Claims (34)

1. A composition for the prevention or treatment of symptoms associated with a gastrointestinal disorder, containing: The types of bacteria that are purified, and where the types of bacteria include: at least one species of bacteria that produces short chain fatty acids (SCFA); And at least one SCFA-metabolizing bacterial species, wherein when the composition is cultured in an acidic environment at pH 6-8 and low oxygen conditions of up to 8 g/l, at least one SCFA-producing bacterial species is present in sufficient quantity to provide an amount of SCFA sufficient to support the growth of at least one species of SCFA-metabolizing bacteria. 2. The composition according to claim 1, characterized in that the SCFA includes acetate, propionate or butyrate. 3. The composition according to claim 1, characterized in that at least one SCFA-producing bacterial species includes Lactobacillus plantarum , Anaerostipes caccae , Faecalibacteruim prausnitzii , Butyricicoccus pullicaecorum , Roseburia inulinivorans , Akkermansia muciniphila or Roseburia hominis . 4. The composition according to claim 1, characterized in that the composition is in the form of a capsule. 5. The composition according to claim 1, wherein the bacterial species include up to 14 bacterial species. 6. The composition according to claim 1, characterized in that the composition contains at least 10 ⁵ colony-forming units of bacteria. 7. The composition according to claim 1, containing from 10 ⁵ to 10 ¹¹ colony-forming units of bacteria. 8. The composition according to claim 1, in which the acidic medium has a pH from 6.15 to 6.40. 9. The composition of claim 1, wherein low oxygen conditions include oxygen availability in the culture medium of up to 8 g/L. 10. A composition for the prevention or treatment of symptoms associated with a gastrointestinal disorder, comprising: species of bacteria that are purified and in suspension, and wherein the species of bacteria includes: at least one species of bacteria that produces short chain fatty acids (SCFA); And at least one SCFA-metabolizing bacterial species, wherein when the composition is cultured in an acidic environment at pH 6-8 and low oxygen conditions of up to 8 g/l, at least one SCFA-producing bacterial species is present in sufficient quantity to provide an amount of SCFA sufficient to support the growth of at least one species of SCFA-metabolizing bacteria. 11. The composition according to claim 10, wherein the suspension is a homogeneous suspension. 12. The composition of claim 10, wherein the SCFA comprises acetate, propionate or butyrate. 13. The composition of claim 10, wherein at least one short-chain fatty acid (SCFA)-producing bacterial species includes Lactobacillus plantarum, Anaerostipes caccae, Faecalibacteruim prausnitzii, Butyricicoccus pullicaecorum, Roseburia inulinivorans, Akkermansia muciniphila or Roseburia hominis . 14. The composition according to claim 10, characterized in that the bacterial species include up to 14 bacterial species. 15. The composition according to claim 10, characterized in that the composition contains at least 10 ⁵ colony-forming units of bacteria. 16. The composition according to claim 10, containing from 10 ⁵ to 10 ¹¹ colony-forming units of bacteria. 17. Composition according to claim 10, in which the acidic medium has a pH from 6.15 to 6.40. 18. The composition of claim 10, wherein low oxygen conditions include oxygen availability in the culture medium of up to 8 g/L. 19. A composition for the prevention or treatment of symptoms associated with a gastrointestinal disorder, characterized in that the composition is in powder form and includes: The types of bacteria that are purified, and where the types of bacteria include: at least one species of bacteria that produces short chain fatty acids (SCFA); And at least one SCFA-metabolizing bacterial species, wherein when the composition is cultured in an acidic environment at pH 6-8 and low oxygen conditions of up to 8 g/l, at least one SCFA-producing bacterial species is present in sufficient quantity to provide an amount of SCFA sufficient to support the growth of at least one species of SCFA-metabolizing bacteria. 20. The composition of claim 19, wherein the SCFA comprises acetate, propionate or butyrate. 21. The composition of claim 19, wherein at least one short-chain fatty acid (SCFA)-producing bacterial species includes Lactobacillus plantarum , Anaerostipes caccae , Faecalibacteruim prausnitzii , Butyricicoccus pullicaecorum , Roseburia inulinivorans , Akkermansia muciniphila or Roseburia hominis . 22. The composition of claim 19, wherein the bacterial species includes up to 14 bacterial species. 23. The composition according to claim 19, characterized in that the composition contains at least 10 ⁵ colony-forming units of bacteria. 24. The composition according to claim 19, containing from 10 ⁵ to 10 ¹¹ colony-forming units of bacteria. 25. Composition according to claim 19, in which the acidic medium has a pH from 6.15 to 6.40. 26. The composition of claim 19, wherein low oxygen conditions include oxygen availability in the culture medium of up to 8 g/L.