SI24543A - Method for the isolation and selection of the bacterial strains, the bacterial strains and the method of their use - Google Patents

Abstract

The present invention describes the process of isolating and selecting new strains of Lactobacillus and Bifidobacterium from bioptic samples of the human intestinal mucosa. The strains were selected on the basis of susceptibility to antimicrobials, viability in simulated gastrointestinal conditions and the demonstration of antimicrobial activity. The basis for this invention are also the entire genomic sequences of Lactobacillus fermentum L930BB and Bifidobacterium animalis subsp. animalis IM386. The invention relates to the use of either of these two strains or both together in the production of a probiotic or pharmaceutical product, in particular in preventing and / or treating functional disorders of the gastrointestinal tract and diseases in the area of the lower digestive tract, such as irritable bowel syndrome (IBS) and chronic inflammatory bowel disease (IBD) (Crohn's disease and ulcerative colitis).

Description

1 • ·

METHOD FOR ISOLATION AND SELECTION OF BACTERIAL STRAINS, BACTERIAL STRAINS AND METHOD OF THEIR USE

PROBLEM

The invention relates to two new bacterial strains from the intestinal mucosa of the colon of a child and their use as probiotics, in particular as immunomudolatoma biotherapeutic agents.

BACKGROUND OF THE INVENTION

The mucous membrane of the gastrointestinal tract in humans is the largest surface on which the interaction between the host and the external environment takes place, so it plays an important role in the defense of the host. It is densely populated with the intestinal microbiota. The intestinal microbiota plays a key role in the functioning of the host’s immune system, affects digestion and metabolism, and is involved in communication between the brain and gut. Thus, it is essential for maintaining normal intestinal physiology and health. Intestinal immune homeostasis is achieved through the interaction between epithelial cells, macrophages, and dendritic cells (DC), which is a response to the intestinal microbiota and is reflected in T cell differentiation. The commensal microbiota influences the development and training of the innate and acquired immune system, regulated by various PRR receptors (pattem recognition receptors) that recognize bacterial molecular patterns of MAMP (microbe-associates molecular pattems). The ability of the host to distinguish between pathogenic and healthy intestinal bacteria, which is crucial for the tolerance of the normal intestinal microbiota, is provided by Toll-like receptors (TLRs), which belong to one of the two classes of PRR that predominate in the gut. Tolerance mechanisms allow the host organism to prevent both infections and tissue damage as a result of an excessive inflammatory response. Disruption of the intestinal microbiota can lead to decreased tolerance to commensal bacteria and to some inflammatory diseases, such as Intestinal Bowel Syndrome (IBS) and Chronic Inflammatory Bowel Disease (IBD).

Immune mechanisms involve the production of cytokines. Peyer's patches are specialized lymphoid aggregates on which M cells are located, arranged throughout the 2 «··· the entire small and large intestine. At these sites, the presentation of luminal antigens, including bacteria, takes place, resulting in stimulation of the corresponding T and B cells, inducing a web of cytokines, and secretion of antibodies into the digestive tract.

The cytokine IL-10 is a typical anti-inflammatory cytokine secreted by Th2, Treg cells, monocytes, and macrophages. IL-10 inhibits the expression of inflammatory cytokines such as TNF-α, IL-1, IL-6, IL-8 and IL-12. Weak production of the anti-inflammatory cytokine IL-10 in the intestinal mucosa of patients with IBD is considered an important factor in the pathogenesis of IBD, so this cytokine is of interest for therapeutic use. In a mouse model of colitis, mice (IL) -10 (- / -) were shown to spontaneously develop intestinal inflammation. Individual probiotic strains, which are characterized by raising the level of IL-10 and lowering the level of the inflammatory cytokine IL-12 in vitro, are able to protect animals from the development of colitis.

Different bacteria can elicit a different immune response in different immune cells, inflammatory or anti-inflammatory, depending on the strain. Different strains of the genera Lactobacillus and Bifidobacteria can also trigger different cytokine responses.

Lactic acid bacteria {Enterococcus, Lactobacillus, Lactococcus, Leuconostoc and Pediococcus) and bifidobacteria have been used in the production of fermented foods for centuries. Today, they are widely used in the food industry, animal feed industry and pharmaceutical industry as a culture starter in fermentation processes and as probiotics.

Probiotics are defined as " living microorganisms that have a beneficial effect on the host's health if ingested in an adequate amount " (FAO / WHO. 2002. Guidelines for the Evaluation of Probiotics in Food, London). The classification of individual strains as probiotics must be based on scientific evidence that exerts a specific (strain-specific) effect on the host.

Lactobacillus fermentum is a Gram-positive heterofermentative species of bacteria of the genus Lactobacillus. This species is most commonly isolated from sourdough or from fermented animal and plant material, but is also often found on the mucosal surface of healthy humans and in breast milk (Jimenez, E. et al. 2010. Journal of Bacteriology. 192: 4800-4800) . This species is included in the list of presumed safe presumption of safety (QPS) biologically active substances recommended for use in 3 • · food and feed, according to the Scientific Council and units of the European Food Safety Authority (EFSA), (2012. EFSA Journal. 10: 3020). So far (until October 2013), the sequences of the entire genomes of the three strains of L. fermentum (http://www.ncbi.nlb.nih.gOv/eenome/711. 10 October 2013) have been published, i.e. CECT 5716, F-6 and IFO 3956. Lactobacillus fermentum CECT 5716 is a human isolated strain having antimicrobial, anti-inflammatory, and immunomudolatome properties (Jimenez, E. et al. 2010. Journal of Bacteriology. 192: 4800-4800). F-6 has been isolated from traditional dairy products produced by minorities in China and has some good probiotic properties such as good resistance to low pH values, bile salts, and antibacterial activity. IFO 3956, an isolate from fermented plant material, produces cobalamin and reuterin (Morita, H. et al. 2008. DNA Research. 15: 151-161).

Some strains of L. fermentum are classified as probiotics and are found in probiotic products. Commercial probiotic strains include L. fermentum PCC and ME-3. Bifidobacterium animalis are Gram-positive anaerobic rod-shaped bacteria that are often found in the colon of most mammals, including humans. The species is included in the list of presumed safe presumption of safety (QPS) biologically active substances recommended for use in food and feed, according to the Scientific Council and units of the European Food Safety Authority (EFSA) (2012. EFSA Journal. 10: 3020) .

Several strains of this species containing a subspecies of B. animalis ssp. lactis in B. animalis ssp. animalis, is used as a probiotic. Examples of the use of this species as probiotics include reducing gastrointestinal inflammation and related diseases such as IBD (US 2012/0308523, US 2008/0107635), reducing abdominal circumference (US 2010/0303763), preventing or treating upper respiratory tract infections ( WO2013104783), effective alleviation of liver damage by alcohol (CN102845528), action against the pathogen Enterobacter sakazakii (KR20120107780), immune stimulation and adaptation of the intestinal microbiota (CN101260377) and synthesis of vitamins BI, B2 and B3 (MD20020020).

To date, the entire genome sequence of one representative of Bif animalis ssp. animalis., i.e. a strain of ATCC 25527 isolated from rat feces (Loquasto, J. R. et al. 2011. Journal of Dairy Science. 94: 5864-5870), and eleven representatives of B. animalis ssp. lactis, including BB-12, a highly prevalent probiotic strain (http://www.ncbi.nlb.nih.gov/genome/844.10 October 2013). 4

Irritable bowel syndrome (IBS) is a functional disorder of the gastrointestinal tract characterized by abdominal pain or discomfort, irregular bowel movements, and constipation or diarrhea. Patients can be divided into three subtypes according to faecal excretion: predominant diarrhea (IBS-D), predominant constipation (IBS-C), or mixed subtype (IBS-M). Symptoms are also accompanied by visceral pain. Some other non-gastrointestinal symptoms, such as fatigue, burning sensation, bloating, anxiety, and depression, have also been commonly identified. Together, they significantly impair the quality of life of patients. The pathophysiology or etiology of IBS is unknown, and no proven effective therapy is available. Among the possible factors involved in the development of IBS are also genetic factors, altered Gl motility and visceral hypersensitivity, psychological stress and disorders or. changes in the composition of the gastrointestinal microbiota, chronic inflammation and bacterial gastroenteritis. Patients with IBS are often treated with moxonidine and its pharmacologically acceptable acid addition salts, which may reduce visceral sensitivity to pain. This use is described in patent US6300336.

Altered gastrointestinal microbiota is regularly observed in patients with IBS (Kassinen, A. et al. 2007. Gastroenterology. 133: 24-33 and Rajilic-Stojanovic, M. et al. 2011. Gastroenterology. 141: 1792-1801). In studies based on the use of culture techniques, lactobacilli and bifidobacteria concentrations were often lower in patients with IBS, while data on coliform bacteria and enterobacteria were conflicting during the studies. The mechanisms are still unclear. Given that the interactions between the host and the microbiota are two-way, it could also be possible that these changes are secondary rather than the cause of the disease process (Quigley, EMM 2011. Current Opinion and Clinical Nutrition and Metabolic Charms. 14: 497- 503). Recent studies with various probiotics show positive results in terms of relieving symptoms, especially abdominal pain and bloating, and stabilizing the intestinal microbiota (Kajander, K. et al. 2008. Alimentary Pharmacology & Therapeutics. 27: 48-57).

There is growing evidence of chronic inflammation of the intestinal mucosa and immune activation in patients with IBS. Elevated plasma / serum plasma levels of inflammatory cytokines such as IL-Ιβ, TNF-α, IL-6 and IL-8 have been confirmed in patients with IBS compared to healthy humans. 5

Therefore, probiotic bacteria that have the ability to have anti-inflammatory activity may be effective in IBS. The immunomodulatory effect has also been attributed to some probiotics. They can affect the production of individual immunoglobulins (IgA) and cytokines (IL-10, TNF-α, IL-6) through interaction with cells of the immune system.

Ulcerative colitis (UC), pouchitis, and Crohn’s disease (CD) are examples of chronic inflammatory bowel disease (IBD) characterized by chronic inflammation in the gut. There is currently no cure for IBD. IBD is treated by reducing inflammation and controlling gastrointestinal symptoms with aminosalicylates, corticosteroids, and immunosuppressive drugs, antibiotics, or biologics. Changes in the composition of the stool and intestinal mucosa microbiota were detected in both cases of IBD, in Crohn's disease and ulcerative colitis. Because the incidence of IBD is associated with disrupted microbiota and inflammation, certain probiotic strains that have immunomodulatory abilities and the ability to alter the intestinal microbiota represent an alternative to IBD treatment.

The use of probiotics for the prevention and treatment of IBS and IBD is presented in various patents. WO2011092261 discloses, for example, the use of Lactobacillus plantarum CECT 7484, Lactobacillus plantarum CECT 7485 and Pediococcus acidilactici CECT 7483 for the treatment of gastrointestinal diseases or conditions such as IBD, IBS or abdominal bloating and bloating. WO2011051760 describes the use of a strain of Bifidobacterium animalis subsp. lactis DN-173010 for the treatment of disorders associated with gastrointestinal inflammation (CD, UC, IBS). W00042168 proposes immunomodulatomes of Bifidobacterium longum infantis UCC35624 and Lactobacillus salivarius UCC 118, mutants thereof or variants thereof for the treatment of inflammatory diseases, in particular gastrointestinal diseases such as IBD and IBS.

A prerequisite for the effectiveness of probiotics in the prevention and treatment of IBS and IBD is the survival of microorganisms in the passage through the upper gastrointestinal tract, which is characterized by low pH (stomach), the presence of bile and pancreatic enzymes (duodenum), to intestinal mucosa and successful intestinal colonization . Probiotic bacteria are often isolated from sludge samples as described in patents WO2011 / 092261, WO2008 / l 17266, WO2012126481 and many others. Bacteria found in the faeces provide survival under conditions typical of the upper digestive system, but these bacteria do not necessarily colonize the intestinal mucosa. It can be assumed that bacteria originating from the intestinal mucosa will have a better chance of colonizing the intestinal mucosa after ingestion as probiotics, as they are better adapted to the intestinal mucosal environment. The use of bacterial isolates from human biopsy specimens has not yet been described in the patent literature. The bacteria described in patent W00042168 are also isolated from the human digestive tract, but compared to the present invention, they are obtained from pieces of intestinal tissue obtained by resection, which requires a different method for isolation and selection of bacterial strains.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes a process for selecting new bacterial strains presumably belonging to the genera Lactobacillus and Bifidobacterium by culturing and purifying on UriSelect 4 agai. coli, Proteus, and Enterococcus, based on differences in bacterial β-galactosidase, tryptophan deaminase, and β-glucosidase activity. To date, UriSelect 4 has not been used to grow or purify bacteria of the genera Lactobacillus and Bifidobacterium. Cultivation on UriSelect 4 allows the differentiation of Lactobacillus and Bifidobacterium strains, which cannot be distinguished on conventional media for these groups of bacteria, such as Rogosa and TOS, on the basis of color and morphology (Example 1, Figure 1). Purification of putative Lactobacillus and Bifidobacterium strains at UriSelect 4 was followed by testing for antibiotic susceptibility to these strains and their ability to survive in the gastrointestinal tract after oral ingestion and to populate human intestinal mucosa, a special feature of selected strains selected from .

The main advantage of the present invention is a method for selecting probiotic microbial strains, comprising the following steps: • obtaining a human intestinal mucosa by biopsy; • isolation of possible probiotic bacterial strains; 7 • · • use of the UriSelect 4 chromogenic medium to distinguish isolates of the genera Lactobacillus and Bifidobacterium; • selection of strains that are important for human use in terms of antimicrobial susceptibility, important in medicine and veterinary medicine; • selection of strains which, after ingestion, are capable of surviving the passage through the gastrointestinal tract to the intestinal mucosa and which are adapted to the human intestinal mucosa and can therefore inhabit it. According to the invention, the intestinal mucosa is preferably of human origin. The test probiotic strains according to the method of the invention can be any probiotic bacteria selected from the genera Lactobacillus and Bifidobacterium. These probiotic strains are preferably obtained from biopsy samples of the intestinal mucosa, even more preferably from human. Further details of the process related to the invention are given below in " Example 1 ".

As described in " Example 4 ", the susceptibility of the bacterial isolates of this invention to selected antimicrobials was determined by E-test (Bio - Merieux, France), as the susceptibility of bacterial strains to antimicrobials important in medicine and veterinary medicine is important. criterion for the selection of bacteria intended for use in humans and animals. EFSA (European Food Safety Authority) criteria, updated in 2012, were used to assess resistance to antibiotics important in medicine and veterinary medicine (ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, chloramphenicol). EFSA Journal. 10: 2740).

As described in " Example 5 " and shown in Figures 8 and 9, the in vitro resistance of the bacterial isolates of this invention under mammalian gastrointestinal conditions (acidic environment, bile salts and pancreatic enzymes) was tested. Tests have shown that the strains of the present invention survive after 3 hours of exposure to simulated gastric juice with pFl 2 or more. The bacteria of the present invention spend 3 hours in a suspension of simulated intestinal juice having a pH of 8 and containing 0.15% bile salts, having previously been exposed to the simulated gastric juice. The term " survival " means that at least 20% of the bacteria originally resuspended in the test medium are determined by a plate counting method known to those skilled in the art and are therefore δ »» • · · · · ··· * • ···· · · · · able to survive the passage through the digestive tract (hereinafter also " GIT "). This is desirable in the case of orally ingested live bacteria, as a prerequisite for colonization of the intestine with the bacteria of the present invention is to survive the passage through the esophagus and stomach.

The strains of the present invention are well adapted to the intestinal mucosa, given that they have been isolated from this microenvironment. This allows them to stay longer in the gut and to interact with the intestinal epithelium and the host’s immune cells. Thus, they can continuously demonstrate their probiotic effects in situ.

In addition, the present invention addresses any bacterial strain selected by the method of the invention. Two of these new bacterial strains, which show a number of properties beneficial to human health, in particular in the prevention or treatment of digestive and other immune-related diseases such as inflammation, were deposited on 24 October 2013 in the Microorganism Collection under the Budapest Agreement. Leibniz-Institut DSMZ-Deutsche Sammlung von Mikro-organisms und Zellkulturen GmbH, Inhoffenstrahe 7 B, 38124 Braunschweig, Germany. This bacterial strain and its characteristics are:

DSMZ 26139: Lactobacillus fermentum L930BB obtained by biopsy of the colon mucosa of a healthy 14-year-old male, selected according to the proposed procedure and labeled with the RAPD profile shown in Figures 2 and 3 and the following genomic sequence (SEK ID N ° 7, SEQ ID N ° 8, SEQ ID N ° 9, SEQ ID N ° 10, SEQ ID N ° 11, SEQ ID N ° 12, SEQ ID N ° 13, SEQ ID N ° 14, SEQ ID N ° 15, SEQ ID N ° 16 , SEQ ID N ° 17, SEQ ID N ° 18, SEQ ID N ° 19, SEQ ID N ° 20, SEQ ID N ° 21, SEQ ID N ° 22, SEQ ID N ° 23, SEQ ID N ° 24, SEQ ID N ° 25, SEQ ID N ° 26, SEQ ID N ° 27, SEQ ID N ° 28, SEQ ID N ° 29, SEQ ID N ° 30, SEQ ID N ° 31, SEQ ID N ° 32, SEQ ID N ° 33, SEQ ID N ° 34, SEQ ID N ° 35, SEQ ID N ° 36, SEQ ID N ° 37, SEQ ID N ° 38, SEQ ID N ° 39, SEQ ID N ° 40, SEQ ID N ° 41 , SEQ ID N ° 42, SEQ ID N ° 43, SEQ ID N ° 44, SEQ ID N ° 45, SEQ ID N ° 46, SEQ ID N ° 47, SEQ ID N ° 48, SEQ ID N ° 49, SEQ ID N ° 50, SEQ ID N ° 51, SEQ ID N ° 52, SEQ ID N ° 53, SEQ ID N ° 54, SEQ ID N ° 55, SEQ ID N ° 56, SEQ ID N ° 57, SEQ ID N ° 58, SEQ ID NO: 59, SEQ ID NO: 6 0, SEQ ID N ° 61, SEQ ID N ° 62, SEQ ID N ° 63, SEQ ID N ° 64, SEQ ID N ° 65, SEQ ID N ° 66, SEQ ID N ° 67, SEQ ID N ° 68, SEQ ID N ° 69, SEQ ID N ° 70, SEQ ID N ° 71, SEQ ID N 72 72, SEQ ID N ° 73, SEQ ID N ° 74, SEQ ID N ° 75, SEQ ID N ° 76, SEQ ID N ° 77, SEQ ID N ° 78) and the properties shown in Figures 6 and 9 and in Tables 1 and 2. In addition to DSMZ 26139, analogues or functionally equivalent strains are also protected. DSMZ 26137: Bifidobacterium animalis ssp. animalis IM386 obtained by biopsy of the colon mucosa of a healthy 14-year-old male, selected according to the proposed procedure and labeled with the RAPD profile shown in Figures 4 and 5 and the following genomic sequence (SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID N ° 81, SEQ ID N ° 82, SEQ ID N ° 83, SEQ ID N ° 84, SEQ ID N ° 85, SEQ ID N ° 86) and the properties shown in Figures 7 and 8 and in Tables 1 and 3 .

In addition to DSMZ 26137, its analogues or functionally equivalent strains are also protected. In addition, by comparing the genomic sequences of the two bacterial strains of the present invention with other bacterial sequences for the same species stored in GenBank, it was found that new specific strains can be selected using the methods of the present invention as described above.

The present invention is based on the determination of the sequence of the entire genome of Lactobacillus fermentum L930BB, which was stored at ENA (European Nucleotide Archive) on 30 October 2013 under serial no. CBUR010000001 to CBUR010000072. The present invention is based on the determination of the sequence of the entire genome of Bifidobacterium animalis subsp. animalis IM386, which was stored at ENA (European Nucleotide Archive) on 24 October 2013 under serial no. CBUQ010000001 to CBUQ010000008.

The subject of the present invention are the nucleotide sequences of SEQ ID N °: 7 to N °: 78 of the genome of Lactobacillus fermentum L930BB and the nucleotide sequences of SEQ ID N °: 79 to SEQ ID N °: 86 of the genome of Bifidobacterium animalis subsp. animalis IM386.

The nucleotide sequences SEQ ID NO: 7 to N °: 78 and SEQ ID NO: 79 to SEQ ID NO: 86 were obtained by sequencing the genomes of Lactobacillus fermentum L930BB and Bifidobacterium animalis subsp. animalis IM386 with Illumina and PacBio systems. Compilation of de novo Illumin readings was performed using Velvet (Zerbino & Bimey, Genome Research 2008 (18): 821-829). Purification of the assembled sequences and assembly of the genome was performed using PacBio data.

The obtained sequence data allow the experts to design and synthesize suitable starting oligonucleotides for amplification of the desired sections by polymerase amplification reaction 10. Accordingly, the present invention also encompasses nucleotide sequences selected from SEQ ID NOs: 7 to N °: 78 and SEQ ID NOs: 79 to SEQ ID NOs: 86, which can be used as starting oligonucleotides to amplify mucleotide sequences.

The invention relates to the use of either of these two strains or both together in the manufacture of a probiotic or pharmaceutical product, in particular in the prevention and / or treatment of functional disorders of the gastrointestinal tract and diseases of the lower gastrointestinal tract, such as irritable bowel syndrome (IBS) and chronic inflammatory bowel syndrome. diseases (IBD) (Crohn's disease and ulcerative colitis). A pharmaceutical product refers to a range of products from medicines to medical devices.

In addition, the present invention contemplates a combination of both strains of the invention. In this regard, the invention relates to biologically pure cultures of each of the strains or a mixture of both. Thus, said aspect of the present invention is the production of various compositions comprising at least one of the strains of the invention, each of the strains being present in a proportion of 0.1% to 99.9%, preferably from 1% to 99%, even more preferably from 10% to 90% . In a preferred embodiment, the composition comprises at least one of the bacterial strains of the invention together with another strain, each of the strains present in the composition in a proportion of 0.1% to 99.9%, preferably from 1% to 99%, even more preferably from 10% to 90%.

A further aspect of the present invention are pharmaceutical compositions comprising at least one strain of the invention, a culture, a composition or a product according to the invention and pharmaceutically acceptable excipients. These pharmaceutical preparations can also be used as nutraceuticals, the excipients being suitable for such a product. The term nutraceutical refers to products ranging from dietary supplements to functional foods and foods for special medical purposes.

In addition, the invention describes a preparation or pharmaceutical preparation for use as a probiotic. In a preferred embodiment, the strains of the present invention are contained in the auxiliary material in an amount of from about 105 ke / g to about 1014 ke / g of auxiliary material (ke means colony units).

A further aspect of the present invention is a composition or pharmaceutical composition for use as a medicament. The required dose of bacterial strains of the invention in the composition or pharmaceutical composition described above varies according to the nature of the disorder or

proposed uses of a composition to be used for either prevention or treatment. By preparing a pharmaceutical composition according to the invention, at least one of the strains is included in a suitable carrier in an amount of from 105 ke / g to about 1014 ke / g of excipient.

However, the activity of new microorganisms in an individual is, of course, dose-dependent. This means that the higher the number of new microorganisms introduced by ingestion or administration of the pharmaceutical preparation or food described above, the higher the protective and / or therapeutic activity of the microorganisms. Because the microorganisms of the present invention are isolated from biopsy specimens of the healthy intestinal mucosa of the child, and because they belong to a species included in the list of presumed safe presumption of safety (QPS) for food and feed, it is considered that even in the largest quantities they can be harmful to the host. Therefore, a large amount of bacteria may be included in a pharmaceutical preparation or in a food product.

Preferably, the strains of the invention, the composition thereof or the pharmaceutical composition should reach the mucosal surfaces. Therefore, they are intended for oral consumption.

Preferably, the strains of the invention, compositions thereof or pharmaceutical compositions are suitable for the treatment or prevention of gastrointestinal disorders and diseases of the lower gastrointestinal tract, such as inflammatory diseases of the gastrointestinal tract. In addition, they can be used in conjunction with other treatments to improve or support their effectiveness.

A further embodiment of the invention is the use of the strains of the invention, preparations thereof or pharmaceutical compositions for the prevention and treatment of chronic inflammatory diseases, such as, but not limited to, chronic inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), due to the ability of probiotic strains. to reduce the production of inflammatory cytokines in activated macrophages (cell model THP-1).

An important aspect of the present invention is therefore a method for preventing or treating a disease, condition or disorder in a mammal, including exposing a mammal, mammalian parts, or mammalian tissue to a strain of Lactobacillus fermentum L930BB, DSMZ deposit no. 26139 or a variant, analogue or functional equivalent thereof. Furthermore, the same is true for Bifidobacterium animalis subsp. animalis IM386 strain, DSMZ deposit no. 26137 or variants, analogues or functional equivalents thereof. 12

Further details of the anti-inflammatory activity of the bacterial strains of the present invention found in vitro on a THP-1 cell model are described below in “Example 6”.

In addition to the properties described above, the fermentation capacity of the strains of the present invention has also been tested because they can improve their settlement in the intestinal mucosa. Carbohydrate metabolism is described in detail in “Example 3”.

A further embodiment of the invention is an edible product consisting of an effective amount of a strain belonging to Lactobacillus fermentum L930BB, its analogues or functional equivalent and / or Bifidobacterium animalis subsp. animalis IM386, its analogue or functional equivalent or a preparation or pharmaceutical preparation thereof, together with appropriate amounts of edible ingredients.

A further aspect of the invention is that the edible product is nutraceutical.

The following examples illustrate the invention. EXAMPLE 1 - Isolation of lactobacilli and bifidobacteria from the intestinal mucosa

The initial isolation was performed from a frozen (at -80 ° C) biopsy sample of the intestinal mucosa of the colon of a 14-year-old male specimen obtained by endoscopy. For bacterial isolation purposes, the tissue was homogenized in 1200 pL of anaerobic diluent with the following composition: 1 g peptone / L; 2 g pig gelatin / L; 8.5 g NaCl / L and 0.557 g cysteine hydrochloride monohydrate / L. The suspension (100 μL) was inoculated onto Rogosa agar (Merck) with 0.05% cysteine (Merck) and on TOS agar (Yakult) with mupirocin (50 mg / L) (Applichem). The plates were incubated anaerobically, using anaerobic vessels and the GENbox system (Bio-Merieux, France) for 72 hours at 37 ° C. The putative Lactobacillus isolates were grown from randomly selected colonies with Rogos medium, on agai MRS with 0.05% cysteine hydrochloride. The putative Bifidobacterium isolates were grown from randomly selected colonies grown on TOS medium. Individual colonies were further purified by repeated sequential culture on UriSelect 4 chromogenic medium (Bio-Rad, France). In the present invention, UriSelect 4 agar was used to differentiate isolates of lactobacilli or bifidobacteria based on the color of the colonies grown on this medium. The color of the colonies depends 13 on the possession of various enzymes that degrade chromogenic substrates in the medium. Gram-positive isolates with morphological characteristics typical of lactobacilli or bifidobacteria were stored in MRS broth with the addition of 0.05% cysteine hydrochloride and 30% glycerol at -80 ° C for further characterization.

Figure 1: Example of using the UriSelect 4 chromogenic medium to differentiate between strains.

The figure shows an example of the use of the UriSelect 4 chromogenic medium to distinguish colonies isolated from human intestinal mucosa during purification of strains. A: The Lactobacillus fermentum strain (L930BB) is seen as colonies in the form of larger circles and the Lactobacillus rhamnosus strain as colonies in the form of smaller circles. B: The Lactobacillus fermentum (L930BB) strain is seen as colonies in the form of larger circles and Bifidobacterium animalis (IM386) as colonies in the form of smaller circles. EXAMPLE 2 - Gene fingerprint (RAPD - PCR) and species identification (16S rDNA sequencing)

RAPD-PCR

Randomly amplified polymorphic DNA (RAPD) analysis was used to differentiate individual isolates. Genomic DNA was prepared by heat treatment of pure culture colonies at 99 ° C for 15 minutes in 10 pL of Go - Taq Flexi buffer (Promega, Madison, WI, USA). Initial oligonucleotides 1254 (SEQ ID NO: 1) and KGT80 (SEQ ID NO: 2) were used for RAPD - PCR. PCR amplification with the initial oligonucleotide KGT80 was performed under the following conditions: 1 cycle at 95 ° C / 3 min, 35 cycles at 95 ° C / 3 min, 37 ° C / 2 min, 72 ° C / 2 min; 1 cycle at 72 ° C / 5 min. Amplification with the initial oligonucleotide 1254 was performed as described in the literature (Hilton A. C. et al. 1996. Journal of Applied Bacteriology. 81: 575-584). PCR samples were analyzed by 2% agarose gel electrophoresis in 5 X TAE buffer for 3.5 h at 70 V. DNA molecular size markers (500 bp and 1000 bp Fermentas molecular scales) were used as a size standard. After electrophoresis, agarose gels were stained with SYBR-safe and examined under a UV transilluminator (Syngene, Cambridge, UK).

Figure 2: RAPD - PCR samples of Lactobacillus bacterial isolates from intestinal mucosal biopsy samples obtained with the initial oligonucleotide 1254.

This figure is an example of the analysis of RAPD - PCR of bacterial isolates from the intestinal mucosa with the initial oligonucleotide 1254, where the amplification products were separated by agarose gel electrophoresis. The L band contains a DNA size marker, and the other bands contain DNA amplifiers of various bacterial isolates. Lane 61 corresponds to strain L930BB of the present invention. A pattern of RAPD line 61 is also observed in some other lines, indicating that these isolates belong to the same strain.

Figure 3: RAPD - PCR samples of Lactobacillus bacterial isolates from intestinal mucosal biopsy samples obtained with the initial oligonucleotide KGT-80.

This figure represents an example of the analysis of RAPD - PCR of bacterial isolates from the intestinal mucosa with the initial oligonucleotide KGT-80, where the amplification products were separated by agarose gel electrophoresis. The L band contains a DNA size marker, and the other bands contain DNA amplifiers of various bacterial isolates. Lane 61 corresponds to strain L930BB of the present invention. A pattern of RAPD line 61 is also observed in some other lines, indicating that these isolates belong to the same strain.

Figure 4: RAPD - PCR samples of Bifidobacterium bacterial isolates from biopsy samples of intestinal mucosa obtained with the initial oligonucleotide 1254.

This figure is an example of the analysis of RAPD - PCR of bacterial isolates from the intestinal mucosa with the initial oligonucleotide 1254, where the amplification products were separated by agarose gel electrophoresis. The L band contains a DNA size marker, and the other bands contain DNA amplifiers of various bacterial isolates. Lane 85 corresponds to strain IM386 of the present invention.

Figure 5: RAPD - PCR samples of bacterial isolates of Bifidobacterium from biopsy samples of intestinal mucosa obtained with the initial oligonucleotide KGT-80. 15 • ·

This figure represents an example of the analysis of RAPD - PCR of bacterial isolates from the intestinal mucosa with the initial oligonucleotide KGT-80, where the amplification products were separated by agarose gel electrophoresis. The L band contains a DNA size marker, and the other bands contain DNA amplifiers of various bacterial isolates. Lane 85 corresponds to strain IM386 of the present invention.

Species identification (16S rDNA sequencing)

Isolates, putative representatives of Lactobacillus and Bifidobacterium, have been identified to a species with partial sequencing of the 16S rRNA gene. Total genomic DNA was extracted from 1 mL overnight culture samples grown in 0.05% cysteine hydrochloride MRS broth at 37 ° C under anaerobic conditions using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA), according to the manufacturer's instructions. Prior to DNA extraction, cells were treated with lysozyme (5 mg / mL) and mutanolysin (25U / mL) in TE buffer. 16S rDNA was amplified using the initial oligonucleotides 27F (SEQ ID NO: 3) and 1495r (SEQ ID NO: 4) in 100 mL of the reaction mixture as described (Yu, J. et al. 2011. Journal of Dairy Science 94: 3229-3241). The amplified DNA was purified by Wizard SV Gel and PCR Clean - Up system according to the manufacturer's instructions (Promega Madison, WI, USA). PCR samples were verified by 1% agarose gel electrophoresis followed by SYBR -Safe staining and UV transilluminatoi examination (Syngene, Cambridge, UK). DNA sequencing was performed in Microsynth (Balgach, Switzerland). To compare nucleotide sequences with sequences in Genbank sequences, the NCBI BLAST 2.2.28 tool (http://blast.ncbi.nlm.nih.gov/blast/) was used: Zhang, Z. et al. 2000. Journal of Computational Biology 7: 203-14).

Result of PCR amplification results obtained by PCR gene amplification for 16S rRNA of strain L930BB with initial oligonucleotides 27F and 1495r (SEQ ID NO: 5):

GGT GCTTGC ACCTG GATTTT ATT GGT AGT CGCC AACG GGCGG AC GGGT GAG T A AC ACGT AGGT A AC CTGCCC AGAAGC GGGGG AC A AC ATTT A GG AG AAC AT GCTAATACCGCATAACANCGTTGTTCGCATGAACAACGCTTAAAAGATGGC TTCTCGCTATCACTTCTGGATGGACCTGCGGTGCATTAGCTTGTTGGTGGGG 16 • ·

TAATGGCCTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGC

CACAATGGGACTGAGACACGGCCCATACTCCTACGGGAGGCAGCAGTAGGG

AATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAG

AAGGGTTTCGGCTC GT A A AGCT CT GTT GTT A AAG A AG AAC ACGT AT G AG AG

TAACTGTTCATACGTTGACGGTATTTAACCAGAAAGTCACGGCTAACTACGT

GCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGG

GCGTAAAGAGAGTGCAGGCGGTTTTCTAAGTCTGATGTGAAAGCCTTCGGC

TTAACCGGAGAAGTGCATCGGAAACTGGATAACTTGAGTGCAGAAGAGGGT

AGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACC

AGTGGCGAAGGCGGCTACCTGGTCTGCAACTGACGCTGAGACTCGAAAGCA

TGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGA

GTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAA

GCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGAC

GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAA

GAACCTTACCAGGTCTTGACATCTTGCGCCAACCCTAGAGATAGGGCGTTTC

CTTCGGGAACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGT

GAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCA

GC ATT AAGTT GGGC ACT CT AGT G AG ACT GCCGGT G AC AA ACCGGAGG AAGG

TGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTG

CTACAATGGACGGTACAACGAGTCGCGAACTCGCGAGGGCAAGCAAATCTC

TTAAAACCGTTCTCAGTTCGGACTGCAGGCTGCAACTCGCCTGCACGAAGTC

GGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGG

CCTTGT AC AC ACCGCCCGT C AC ACC AT G AG AGTTT GT AAC ACCC AAAGTCG

GTGGG

Molecular identification of the bacterial isolate L930BB based on 16S rRNA gene sequence showed the highest homology (99% match, 0% gap) with strains belonging to the species Lactobacillus fermentum, including type strain ATCC 14931.

Sequence of PCR amplification results obtained by PCR amplification of the 16S rRNA gene of the IM386 strain with the initial oligonucleotides 27F and 1495r (SEQ ID NO: 6): 17

CAGTCGAACGGGATCCCTGGCGGCCTGGCTGCCGGGGTGAGAGTGGCGAAC

GGGT G AGT AATGCGT GACC AACCT GCCCTGT GC ACCGGAAT AGCT CCT GGA

AACGGGTGGT A ATACCGGAT GCTCCGCCCC ACCGC ATGGT GGGGT GGGAAA

TGCTTTTTGCGGCATGGGATGGGGTCGCGTCCTATCAGCTTGTTGGCGGGGT

GATGGCCCACCAAGGCGTTGACGGGTAGCCGGCCTGAGAGGGTGACCGGCC

ACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGA

ATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGCGGGATGG

AGGCCTTCGGGTTGTAAACCGCTTTTGTTCAAGGGCAAGGCACGGCTTCGG

GCCGTGTTGAGTGGATTGTTCGAATAAGCACCGGCTAACTACGTGCCAGCA

GCCGCGGT AAT ACGT AGGGTGCG AGCGTT ATCCGG ATTT ATTGGGCGT AAA

GGGCTCGT AGGCGGTTCGTCGCGTCCGGTGT GAAAGTCC ATCGCCT AACGG

TGGATCTGCGCCGGGTACGGGCGGGCTGGAGTGCGGTAGGGGAGACTGGA

ATTCCCGGT GT AACGGT GGAAT GTGT AGAT ATCGGGAAG AAC ACC AAT GGC

GAAGGCAGGTCTCTGGGCCGTTACTGACGCTGAGGAGCGAAAGCGTGGGGA

GCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTG

GATGTGGGGCCCTTTCCACGGGTCCTGTGTCGGAGCCAACGCGTTAAGCATC

CCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGG

GCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAAC

CTTACCTGGGCTTGACATGTGCCGGATCGCCCCGGAAACGGGGTTTCCCTTC

GGGGC CGGTT C AC AGGT GGT GC AT GGT C GTCGT C AGCTCGT GTCGT GAGAT

GTT GGGTT A AGT CCCGC AACG AGCGC AACCCT CGCCGC AT GTT GCC AGCGG

GTTATGCCGGGAACTCATGTGGGACCGCCGGGGTCAACTCGGAGGAAGGTG

GGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTA

CAATGGCCGGTACAACGCGATGCGACACGGTGACGTGGGGCGGATCGCTGA

AAACCGGTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGGCGG

AGTCGCTAGTAATCGCGGATCAGCAACGCCGCGGTGAATGCGTTCCCGGGC

CTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGTAGCACCCGAAGCCGG

TGGCCCGACCCTCGT

Molecular identification of the bacterial isolate IM386 based on the 16S rRNA gene sequence showed the highest homology (100% match, 0% gap) with strains belonging to the subspecies Bifidobacterium animalis subsp. animalis, including type strain ATCC 25527.

Sequencing of genomes of L930BB and IM386 isolates

To determine the genomic nucleotide sequence of L930BB and IM386 isolates, genomic DNA was extracted from 1 mL of overnight culture samples grown in MRS broth with 0.05% cysteine hydrochloride at 37 ° C under anaerobic conditions using the DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Prior to DNA extraction, cells were treated with lysozyme (5 mg / mL) and mutanolysin (25U / mL) in TE buffer. The nucleotide sequence of genomic DNA was obtained by Illumina MySeq, in a "paired end" manner (analyzes performed by the Fundacion Parque Cientifico de Madrid, Madrid, Spain), and by PacBio RS (analyzes performed by Expression Analyzes - A Quintiles Company, Durham, NC 2771, USA). Sequences were compiled using Velvet (Zerbino, D.R. et al. 2008. Genome Research. 18: 821-829; performed by Era7, Granada, Spain). The contigs (72) of the L930BB strain are contained in the attached nucleotide sequence list, which was filed together with the patent application under serial numbers SEQ ID NO: 7 - SEQ ID NO: 78. The contigs (8) of the IM386 strain are in the attached nucleotide sequence list. , filed together with the patent application under serial numbers SEQ ID NO: 79 - SEQ ID NO: 86. EXAMPLE 3 - Morphology and fermentation capacity

Morphology of colonies and cells

Morphological characteristics of colonies and bacterial cells of Lactobacillus fermentum L930BB were described after growth of pure culture for 48 hours on MRS agar with 0.05% cysteine hydrochloride at 37 ° C under anaerobic conditions. The colonies are white, dull, with blurred edges. Gram staining showed Gram-positive, long rod-shaped bacteria (Fig. 6). 19

Morphological characteristics of colonies and bacterial cells of Bifidobacterium animalis IM386 were described after growth of pure culture for 48 hours on MRS agar with 0.05% cysteine hydrochloride at 37 ° C under anaerobic conditions. The colonies are white, shiny, with sharp edges. Gram staining showed Gram-positive rod-shaped bacteria, often organized in pairs or in a V-shape (Figure 7).

Figure 6: Microscopic image of Lactobacillus fermentum L930BB after Gram staining (1000x magnification). This figure shows the micromorphological characteristics of the bacterial cells of Lactobacillus fermentum L930BB of the present invention, visible on microscopic examination of Gram-stained pure cultures. Bacteria of this strain are Gram-positive rod-shaped bacteria that appear as single cells or organized in short chains.

Figure 7: Microscopic image of Bifidobacterium animalis IM386 by Gram stain (1000x magnification). This picture represents the micromorphological properties of the bacterial cells of Bifidobacterium animalis subsp. animalis IM386 of the present invention, visible on microscopic examination of Gram-stained pure cultures. Bacteria of this strain are Gram-positive rods, which are most often organized in V-shaped pairs.

Carbohydrate Metabolism (API)

Analysis of carbohydrate metabolism was performed using a standardized API 50 CH system (Biomerieux, France) consisting of 50 cells containing various substrates from the carbohydrate family and their derivatives (heterosides, polyalcohols, uranium acids). Substrates in lyophilized form were rehydrated with API 50 CHL medium and inoculated with test bacterial strains. During incubation, a change in the color of the indicator dye in the cell is detected as a result of anaerobic acid production and a decrease in pH. The first cell, which does not contain substrate, serves as a negative control.

The strains were grown in a medium adapted for their growth (lactobacilli-MRS, bifidobacteria-MRS with 0.05% cysteine w / v). Colonies were transferred using a cotton swab to API 50 CHL medium. The results were read after a certain incubation time (24h, 48h), depending on the type of reaction. The results are presented in Table 1. • · • · · ·

Lactobacillus fermentum L930BB is able to ferment the following substrates: L-arabinose, D-ribose, D-galactose, D-fructose, D-mannitol, N-acetylglucosamine, esculin, salicin, D-maltose, D-lactose, D-melibiose, D -sucrose, D-trehalose and D-raffinose.

Bifidobacterium animalis subsp. animalis IM386 is able to ferment the following substrates: D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, N-acetylglucosamine, amygdalin, arbutin, esculin, salicin, D-celiobiose, D- maltose, D-lactose, D-trehalose, D-melesitosis, gentiobiose and D-tagatose.

The ability of the bacterial strains of the present invention to ferment various carbohydrates allows them to be used by the probiotic strain in the gut as a carbon source and consequently the efficiency of their colonization is better.

Table 1: Fermentation samples of the strains of the invention. Substrates fermented by the test strain are denoted by +; - negative,? questionable result.

Cell Test Substrate Quantity (mg / cell) Lactobacillus fermentum L930BB Bifidobacterium animalis subsp. animalis IM386 0 CONTROL - - - 1 GLY glycerol 1.64 - - 2 ERY erythritol 1.44 - - 3 DARA D-arabinose 1,4 - - 4 LARA L-arabinose 1,4 + - 5 RIB D-ribose 1, 4 + + 6 DXYL D-xylose 1,4 - - 7 LXYL L-xylose 1,4 - - 8 ADO D-adonitol 1,36 - - 9 MDX methyl-PD-xylopyranoside 1,28 - - 10 GAL D-galactose 1.4 + + 11 GLU D-glucose 1.56 + + 21 • · FRU D-fructose 1.4 + + MNE D-mannose 1.4? + SBE L-sorbose 1,4 - - RHA L-rhamnose 1,36 - - DUL dulcitol 1,36 - - INO inositol 1,4 - - MAN D-mannitol 1,36 + + SOR D-sorbitol 1,36? - MDM methyl-aD- 1.28 - - mannopyranoside MDG methyl-aD- 1.28 - - glucopyranoside NAG N-acetylglucosamine 1.28 + + ΑΜΥ amygdalin 1.08 - + ARB arbutin 1.08? + ESC esculin iron citrate 1.16 + + SAL salicin 1.04 + + CEL D-celiobiose 1.32 - + MAL D-maltose 1.4 + + LAC D-lactose (bovine 1.4 + + source) MEL D -melibiose 1.32 + - SAC D-sucrose 1.32 + - TRE D-trehalose 1.32 + + INU inulin 1.28 - - MLZ D-melesitose 1.32 - + RAF D-raffinose 1.56 + - AMD Amidon (starch) 1.28 - - GLYG glycogen 1.28 - - XLT xylitol 1,4 - - GEN gentiobiose 0.5 - + TUR D-turanose 1.32 - - 41 LYX D-lyxose 1.4 - - 42 TAG D-tagatose 1,4 - + 43 DFUC D-fucose 1,28 - - 44 LFUC L-fucose 1,28 - - 45 DARL D-arabitol 1,4 - - 46 LARL L-arabitol 1,4 - - 47 GNT On gluconate 1.84? ? 48 2KG To 2-Ketogluconate 2.12 - - 49 5 KG To 5-Keto gluconate 1.8 + - EXAMPLE 4 - Susceptibility to antimicrobial substances important in medicine and veterinary E-test (Bio-Merieux, France) to determine sensitivity for antibiotics was performed as described by M. Danielsen and A. Wind (2003. International Journal of Food Microbiology. 82: 1-11), with the following media: a) 90% agar Iso - sensitest, 10% MRS, 0 , 05% cysteine hydrochloride, pH = 6.7 or b) 90% Mueller Hinton agar, 10% MRS, 0.05% cysteine hydrochloride, pH = 6.7 (Klare, I. et al. 2005.Applied and Environmental Microbiology .71: 8982-8986). After 48 hours of incubation in an anaerobic atmosphere (Anaerobox, Bio-Merieux, France), MIC values (minimum inhibitory concentration) were read as recommended by the manufacturer. The EFSA criteria for the assessment of resistance to antibiotics relevant to medicine and veterinary, updated in 2012, were taken into account (2012. EFSA Journal. 10: 2740).

Table 2: Susceptibility of Bifidobacterium animalis subsp. animalis IM386 of the present invention for 9 antimicrobials (mg / L)

Antimicrobial substance EFSA limit MIC (MH) MIC (ISO) value for

MIC (concentration range) • · mm 23 ·· · · ·· ·· ampicillin (AM) (0.016-256) 0.016 0.016 2 vancomycin (VA) (0.016-256) 0.38 0.19 2 gentamicin (GM) 0.064-1024) 64 32 64 kanamycin (KM) (0.016-256) > 256 > 256 nr streptomycin (SM) (0.064-1024) 48 12 128 erythromycin (EM) (0.016-256) 0.125 0.094 1 clindamycin (CM) (0.016-256) 1 0.50 1 tetracycline (TC) (0.016-256) 0.50 0.25 8 chloramphenicol (CL) (0.016-256) 0.75 0.50 4 MIC - minimum inhibitory concentration, MH - 90% Mueller Hinton agar + 10% MRS agar + 0.05% cysteine hydrochloride; ISO - 90% Iso-sensitest + 10% agar MRS + 0.05% cysteine hydrochloride, n.r.-test not required by EFSA; strains with a MIC greater than the cut-off value (2012. EFSA Journal. 10: 2740) are considered resistant. Table 3: Sensitivity of Lactobacillus fermentum L930BB of the present invention to 9 antimicrobials (mg / L) Antimicrobial substance EFSA limit MIC (MH) MIC (ISO) value for (concentration range) MIC ampicillin (AM) (0.016-256) 0.094 0.125 2 vancomycin (VA) (0.016-256) > 256 > 256 no gentamicin (GM) (0.064-1024) 4 1 16 kanamycin (KM) (0.016-256) 32 32 32 streptomycin (SM) (0.064-1024) 12 12 64 ··· · · · · · • ··· · Erythromycin (EM) (0.016-256) 0.50 0.50 1 clindamycin (CM) (0.016-256) 0.016 0.016 1 tetracycline (· · · · • · · · · · · · · · · · · · · TC) (0.016-256) 1 1.5 8 chloramphenicol (CL) (0.016-256) 1.5 1 4 MIC - minimum inhibitory concentration, MH - 90% agar Mueller Hinton + 10% agar MRS + 0.05% cysteine hydrochloride; ISO - 90% Iso-sensitest + 10% agar MRS + 0.05% cysteine hydrochloride, n.r.-test not required by EFSA; strains with a MIC greater than the cut-off value (2012. EFSA Journal. 10: 2740) are considered resistant.

For strains of Bifidobacterium animalis subsp. animalis IM386 and Lactobacillus fermentum L930BB of the present invention were found to be susceptible to nine antimicrobial substances relevant to medicine and veterinary medicine, which EFSA requires to be included in the safety assessment of bacterial strains intended for use in humans and animals. EXAMPLE 5 - Survival in gastrointestinal tract (GIT) conditions

Lactobacillus and Bifidobacterium isolates were incubated in MRS broth with 0.05% cysteine (w / v) for 18 h at 37 ° C under anaerobic conditions. After washing in sterile saline (NaCl, 0.9%) and centrifugation, 50 mL of simulated gastric juice was added to the bacterial cells, with the following composition: NaCl, 125 mmol / L; KCl 7 mmol / L; NaHCO 3, 45 mmol / L and pepsin, 3 g / L. The final pH was adjusted with HCl to pH 2 and 3, or with NaOH to pH 7. The bacterial suspension was incubated by shaking (200 rpm), simulating peristalsis, at 37 ° C. Aliquots for determining the number of colony units were taken after 0, 90 and 180 min. Simulated intestinal juice was prepared from 0.1% (w / v) pancreatin and 0.15% (w / v) bile salts of Oxgall in distilled water, and the pH was adjusted to 8 with NaOH. The bacterial suspension was incubated for a further 180 min, and after 0, 90, and 180 min, samples were collected to count colony units (Femandez, MF 2003. Journal of Applied Microbiology. 94: 449-455). 25 25 • · ·

Survival results of Lactobacillus fermentum L930BB, Bifidobacterium animalis subsp. animalis IM386 and the reference probiotic strain Lactobacillus rhamnosus GG under gastrointestinal conditions are presented in Figures 8 to 10.

Survival of Lactobacillus fermentum L930BB and Bifidobacterium animalis subsp. animalis IM386 in simulated gastric juice is 82-91% and 74-83%, depending on the pH of gastric juice. Particularly under very acidic conditions (pH = 2), the survival of both strains of this invention was better than the survival of the reference probiotic strain Lactobacillus rhamnosus GG (< 1%).

Survival of Lactobacillus fermentum L930BB and Bifidobacterium animalis subsp. animalis IM386 in the simulated intestinal juice were 21–81% and 26–100%, depending on the pH of the gastric juice to which the cells were exposed prior to treatment in the intestinal juice. Especially for bacteria exposed to intestinal juice after treatment with gastric juice (pH = 3), the survival of both strains of this invention was better than the survival of the reference probiotic strain Lactobacillus rhamnosus GG (1.4%).

Figure 8: Survival of Bifidobacterium animalis subsp. animalis IM386 in simulated gastrointestinal conditions.

This figure shows the survival of the IM386 strain of this invention in simulated gastric juice (pH = 2, pH = 3, pH = 7) and simulated intestinal juice (pH = 8). After exposure to the simulated gastric juice for 180 min, the bacterial cells were transferred to the simulated intestinal juice and incubated for the next 180 minutes. The results are presented as the average of three independent experiments.

Figure 9: Survival of Lactobacillus fermentum L930BB in simulated gastrointestinal conditions.

This figure shows the survival of the L930BB strain of the present invention in simulated gastric juice (pH = 2, pH = 3, pH = 7) and simulated intestinal juice (pH = 8). After exposure to the simulated gastric juice for 180 min, the bacterial cells were transferred to the simulated intestinal juice and incubated for the next 180 minutes. The results are presented as the average of three independent experiments.

Figure 10: Survival of Lactobacillus rhamnosus GG in simulated gastrointestinal conditions. 26 26

«· 9 m

This figure shows the survival of the reference probiotic strain GG in simulated gastric juice (pH = 2, pH = 3, pH = 7) and simulated intestinal juice (pH = 8). After exposure to the simulated gastric juice for 180 min, the bacterial cells were transferred to the simulated intestinal juice and incubated for the next 180 minutes. The results are presented as the average of three independent experiments. EXAMPLE 6 - Anti-inflammatory activity

To investigate the anti-inflammatory potential of Lactobacillus and Bifidobacterium strains, we measured the production of TNF-α, IL-8, IL-10 and IL-6 in THP-1 cells (ATCC TIB - 202, human monocyte cell line) after stimulation with LPS (lipopolysaccharides). E. coli Olll: B4), by bacteria or by a combination of LPS and bacteria. Pure Lactobacillus and Bifidobacterium strains were cultured in MRS with 0.05% L -cysteine hydrochloride under anaerobic conditions for 18 hours. All bacteria were removed by centrifugation and washed with phosphate buffer (PBS). The number of colony units (k.e.) was determined by counting on plates, on MRS medium with 0.05% L - cysteine hydrochloride.

THP-1 cells were grown in RPMI medium supplemented with FBS, glucose, L-glutamine, and antibiotics and resuspended at a concentration of 106 cells / mL in the same medium on 24-well cell culture plates. Prior to the experiment, cells were stimulated with PMA (phorbol ester) for 48 hours to induce differentiation into macrophages. The cells were then incubated with either the selected bacterial strain {Lactobacillus fermentum L930BB, Bifidobacterium animalis subsp. animalis IM386), LPS or a combination of LPS and bacterial resuspension in complete RPMI medium, without antibiotics. The plates were incubated at 37 ° C in a 5% CO2 atmosphere. All tests were performed in triplicate. Samples of cell line supernatants collected after 4, 6 and 8 hours of incubation were analyzed for the content of cytokines IL-10, IL-6, IL-8 and TNF-α using a standard ELISA (Enzyme - Linked Immunoabsorbent test, eBioscience) , USA), according to the manufacturer's instructions. The technique is well known to those skilled in the art. The results obtained (Table 4) indicate that the consumption of the probiotic strains of this invention could have a beneficial effect on some inflammatory diseases of the host. Lactobacillus fermentum L930BB induced IL-10 production in THP-1 cells. LPS alone did not induce IL-10 production.

Lactobacillus fermentum L930BB reduced LPS-induced production of pro-inflammatory IL-6. Bifidobacterium animalis subsp. animalis IM386 reduced LPS-induced production of pro-inflammatory IL-6, IL-8, and TNF-α. Bifidobacterium animalis subsp. animalis IM386 and Lactobacillus fermentum L930BB added together reduced the production of LPS-induced pro-inflammatory IL-6. Bifidobacterium animalis IM386 and Lactobacillus fermentum L930BB added together reduced the production of pro-inflammatory IL-6 induced by LPS.

Table 4: Effect of Lactobacillus fermentum L930BB and Bifidobacterium animalis subsp. animalis IM386 on the production of the cytokines interleukin 6, interleukin 8, TNF-α and interleukin 10 in LPS-induced THP-1 cells during co-incubation.

Interleukin 6 (IL-6) 1 Interleukin 8 (IL-8) 1 TNF-α 1 Interleukin 10 (IL-10) 2 IM 386 Jr 8 hours 99% ± 1% and 8 hours 37% ± 8% and 8 hours 91 % ± 1% No increase L 930 BB i 6 hours 74% ± 1% No decrease No decrease t 8 hours 812% ± 18% IM 386 + L 930 BB -2 -: - 1 i 8 hours 93% ± 1% i 6 hours 37% ± 19% No decrease No increase in the% increase in the amount of IL-10 produced in THP-1 cells during co-incubation with the isolates of this invention (IM386, L930BB or a combination of both) compared to the amount of IL-10 produced in the cells THP-1 induced by LPS only. 1% reduction in the amount of cytokines (IL-6, IL-8 or TNF-α) produced in LPS-induced THP-1 cells during co-incubation with the isolates of this invention (IM386, L930BB or a combination of both) compared to the amount cytokines produced in LPS-only THP-1 cells. 28 Time (6 hours or 8 hours) indicates the sampling time at which the greatest effect of bacterial strains on cytokine production in THP-1 cells was observed.

Claims (20)

29 A process for the isolation and selection of bacterial strains from biopsy samples of the human intestinal mucosa, comprising the steps of: (a) obtaining strains from the human intestinal mucosa; (b) isolation of any bacterial strains; (c) use of the UriSelect 4 chromogenic medium to distinguish isolates of bacterial strains; (d) the selection of potential strains which are presumed to be susceptible to antimicrobials relevant to mammals; (e) the selection of possible strains which are presumed to survive in mammalian gastrointestinal conditions. The method of claim 1, wherein the potential bacterial strain is of the genus Lactobacillus or Bifidobacterium. 3. Lactobacillus fermentum strain L930BB, stored in DSMZ, deposit no. 26139, its analogues or functionally equivalent strains. A strain of Lactobacillus fermentum L930BB characterized by a genomic sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO. ° 12, SEQ ID N ° 13, SEQ ID N ° 14, SEQ ID N ° 15, SEQ ID N ° 16, SEQ ID N ° 17, SEQ ID N ° 18, SEQ ID N ° 19, SEQ ID N ° 20 , SEQ ID N ° 21, SEQ ID N ° 22, SEQ ID N ° 23, SEQ ID N ° 24, SEQ ID N ° 25, SEQ ID N ° 26, SEQ ID N ° 27, SEQ ID N ° 28, SEQ ID N ° 29, SEQ ID N ° 30, SEQ ID N ° 31, SEQ ID N ° 32, SEQ ID N ° 33, SEQ ID N ° 34, SEQ ID N ° 35, SEQ ID N ° 36, SEQ ID N ° 37, SEQ ID N ° 38, SEQ ID N ° 39, SEQ ID N ° 40, SEQ ID N ° 41, SEQ ID N ° 42, SEQ ID N ° 43, SEQ ID N ° 44, SEQ ID N ° 45 , SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 30 49, SEQ ID N ° 50, SEQ ID N ° 51, SEQ ID N ° 52, SEQ ID N ° 53, SEQ ID N ° 54, SEQ ID N ° 55, SEQ ID N ° 56, SEQ ID N ° 57, SEQ ID N ° 58, SEQ ID N ° 59, SEQ ID N ° 60, SEQ ID N ° 61, SEQ ID N ° 62, SEQ ID N ° 63, SEQ ID N ° 64, SEQ ID N ° 65, SEQ ID N ° 66, SEQ ID N ° 67, SEQ ID N ° 68, SEQ ID N ° 69, SEQ ID N ° 70, SEQ ID N ° 71, SEQ ID N ° 72, SEQ ID N ° 73, SEQ ID N ° 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78. Lactobacillus enzyme strain L930BB, according to any one of claims 3 to 4, having anti-inflammatory activity. 6. Lactobacillus fermentom strain L930BB, DSMZ deposit no. 26139 or variants, analogs or functional equivalents thereof for use in the prevention and / or treatment of a disease, condition or disorder in a mammal, wherein the use comprises exposing the mammal, a portion of the mammal or mammalian tissue to said strain. 7. Strain Bifidobacterium animalis subsp. animalis IM386, stored in DSMZ, deposit no. 26137, its analogues or functionally equivalent strains. 8. Bifidobacterium animalis subsp. animalis IM386 characterized by a genomic sequence selected from the group consisting of SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID N ° 85, SEQ ID N ° 86. 9. Bifidobacterium animalis subsp. animalis IM386 according to any one of claims 7 to 8, having anti-inflammatory activity. 10. Strain Bifidobacterium animalis subsp. animalis IM386, DSMZ deposit no. 26137 or variants, analogs or functional equivalents thereof for use in the prevention and / or treatment of a disease, condition or disorder in a mammal, wherein the use comprises exposing the mammal, part of the mammal or mammalian tissue to said strain. 31 Use of a strain according to any one of claims 3 to 5 or 7 to 9 for the preparation of a probiotic as a nutraceutical and / or pharmaceutical product, in particular for the prevention and / or treatment of functional disorders of the gastrointestinal tract and diseases in the lower gastrointestinal tract. 12. Pharmaceutical preparation containing at least a strain of Lactobacillus fermentum L930BB, stored in DSMZ, deposit no. 26139, a culture and a pharmaceutically acceptable excipient. 13. A pharmaceutical preparation containing at least Bifidobacterium animalis subsp. animalis IM386, stored in DSMZ, deposit no. 26137, a culture and a pharmaceutically acceptable excipient. The preparation contains at least one of the strains according to any one of claims 3 to 5 or 7 to 9, and wherein each of the bacterial strains is present in the preparation in a ratio of from 0.1% to 99.9%, preferably from 1% up to 99%, even more preferably from 10% to 90%. A preparation according to any one of claims 12 to 14 for use as a probiotic. A preparation according to any one of claims 12 to 14 for use as a medicament. A strain according to any one of claims 3 to 5 or 7 to 9, or a preparation according to any one of claims 11 to 16 for use in the treatment and / or prevention of functional disorders of the gastrointestinal tract and diseases in the lower gastrointestinal tract. A strain according to any one of claims 3 to 5 or 7 to 9, or a preparation according to any one of claims 11 to 16 for use in the treatment and / or prevention of irritable bowel syndrome and chronic inflammatory bowel disease. 32 An edible product comprising an effective amount of a strain according to any one of claims 3 to 5 or 7 to 9 or a preparation according to any one of claims 12 to 16, together with corresponding amounts of other edible ingredients. The edible product of claim 19, which is a nutraceutical.