probiotic bifidobacterial strain

专利摘要:
PROBIOTIC BIFIDOBACTERIA TREE.The present invention relates to a strain of probiotic bifidobacterium AH1714 which is significantly immunomodulatory after oral consumption. The strain is useful as an immunomodulatory biotherapeutic agent.
公开号:BR112012011315A2
申请号:R112012011315-1
申请日:2010-11-11
公开日:2020-08-25
发明作者:Liam O'Mahony；Barry Kiely；John Francis Cryan；Timothy Dinan；Eileen Frances Murphy
申请人:Alimentary Health Limited；The Procter & Gamble Company；
IPC主号:

专利说明:

'Invention Patent Descriptive Report for "CEPA DE B / - - PROBIOTIC FIDOBACTERIA".
INTRODUCTION 2 The present invention relates to a strain of bifidobacteria and its use as a probiotic bacterium, in particular as an immunomodulatory biotherapeutic agent.
The defense mechanisms to protect the human gastrointestinal tract against colonization by intestinal bacteria are highly complex and involve immunological and non-immunological aspects (1). The innate defense mechanisms include low stomach pH, bile salts, peristalsis, mucin layers and anti-microbial compounds, such as' lysozyme (2). Immunological mechanisms include es- lymphoid aggregates. specialized, underlying M cells, called peyers' plates, which are distributed throughout the small intestine and colon (3). Lumen antigens present in these sites result in the stimulation of suitable subsets of T and B cells with the establishment of cytokine networks and the secretion of antibodies to the gastrointestinal tract (4). In addition, the presentation of the antigen can occur through epithelial cells to the epithelial lymphocytes and to the underlying lamina propria immune cells (5).
Therefore, the host invests substantially in the immune defense of the gastrointestinal tract (TGI). However, due to the gastrointestinal mucosa being the largest surface on wed! the host interacts with the external environment, there must be specific control mechanisms to regulate the immune response capacity to the 90.718 kg (100 tons) of food that is managed by the gastrointestinal tract during the average life span. In addition, the gastrointestinal tract is colonized by more than 500 species of bacteria, reaching 10 "to 10 ' / G in the colon. Thus, these control mechanisms must be able to distinguish non-pathogenic adherent bacteria from invasive pathogens, which could cause significant damage to the host.In fact, the intestinal flora contributes to the defense of the host, competing with the potentially pathogenic microorganisms recently ingested.
'The bacteria present in the human gastrointestinal tract can. cause inflammation. Aberrant immune responses to native microflora 'have been involved in certain unhealthy states, such as the intestinal inflammatory process. Antigens associated with the normal general flora- —release immunological tolerance, and failure to achieve this tolerance is an important mechanism of mucosal inflammation (6). The evidence for this failure in tolerance includes an increase in the levels of antibodies directed against the flora of the gastrointestinal tract in patients with inflammatory bowel disease (IVD). The present invention is directed towards a strain of bifidobacteria that has been shown to have immunomodulatory effects by modulating cytokine levels or antagonizing and excluding pro-microorganisms. inflammatory diseases of the gastrointestinal tract.
DECLARATIONS OF THE INVENTION The invention features an isolated strain of NCIMB bifidobacterium
41676.
The bifidobacterial strain may be in the form of viable cells. The bifidobacterial strain may be in the form of non-viable cells. Bifidobacteria can be isolated from the tissue for colonic biopsy of a healthy human individual. The bifidobacterial strain can be significantly immunomodulatory after oral consumption in humans.
The invention also provides a formulation that comprises a bifidobacterial strain as described in the present invention. The formulation may also comprise a probiotic material. The formulation may further comprise a prebiotic material. The formulation may further comprise an ingestible vehicle. The ingestible vehicle can be a pharmaceutically acceptable vehicle, such as a capsule, tablet, or powder. The ingestible vehicle can be a food product such as acidified milk, yogurt, frozen yogurt, powdered milk, milk concentrate, curd, sauces or drinks.
The formulation can further comprise a protein and / or peptide, in particular proteins and / or peptides that are rich in glutamine / glutamate, a lipid, a carbohydrate, a vitamin, mineral and / or microelement. The strain
'of bifidobacteria can be present in an amount of more than 10º cfu. per gram of the formula. The formulation may further comprise an adjuvant. The formulation may further comprise a bacterial component. The formulation can also comprise a drug entity. The formulation may also comprise a biological compound. The formulation can be used for immunization and vaccination protocols.
The invention also provides a strain of bifidobacterium or a formulation, as described here, for use in food products.
The invention also provides a bifidobacterial strain or formulation, as described here, for use as a medicament.
The invention also provides a bifidobacterial strain or formulation, as described herein, for use in prophylaxis and / or treatment. of undesirable inflammatory activity.
The invention also provides a bifidobacterial strain or a formulation, as described here, for use in the prophylaxis and / or treatment of undesirable gastrointestinal inflammatory activity, such as inflammatory bowel disease, for example: Crohn's disease or ulcerative colitis, bowel syndrome irritable; bursitis; or post-infectious colitis.
The invention also provides a bifidobacterial strain or a formulation, as described here, for use in the prophylaxis and / or treatment of gastrointestinal cancer.
The invention also provides a bifidobacterial strain or a formulation, as described here, for use in the prophylaxis and / or treatment of systemic disease, such as rheumatoid arthritis.
The invention also provides a bifidobacterial strain or a formulation, as described here, for use in the prophylaxis and / or treatment of autoimmune disorders due to undesirable inflammatory activity.
The invention also provides a bifidobacterial strain or a formulation, as described herein, for use in the prophylaxis and / or treatment of cancer due to undesirable inflammatory activity.
The invention also provides a bifidobacterial strain or a formulation, as described here, for use in cancer prophylaxis.
'The invention also provides a bifidobacterial strain or a' formulation, as described here, for use in the prophylaxis and / or treatment] of diarrheal disease due to undesirable inflammatory activity, such as diarrhea associated with Clostridium diffícile, diarrhea associated with rotavirus or post-infectious diarrhea, or diarrheal disease due to an infectious agent, such as E. coli.
The invention also provides a bifidobacterial strain or a formulation, as described herein, for use in the preparation of bio-therapeutic anti-inflammatory agents for the prophylaxis and / or treatment of undesirable inflammatory activity. Bifidobacterial strains, as described here, can be used in the preparation of a panel of biotherapeutic agents for modi. fication of IL-10 levels.
The invention also provides a bifidobacterial strain or a formulation, as described here, for use in the prevention and / or treatment of inflammatory disorders, immunodeficiency, inflammatory bowel disease, irritable bowel syndrome, cancer (particularly of the gastrointestinal and immune systems) , diarrheal disease, antibiotic-associated diarrhea, pediatric diarrhea, appendicitis, autoimmune disorders, multiple sclerosis, Alzheimer's disease, rheumatoid arthritis, celiac disease, diabetes mellitus, organ transplantation, bacterial infections, viral infections, fungal infections periodontal disease, urogenital disease, sexually transmitted disease, HIV infection, HIV replication, HIV-associated diarrhea, surgery-associated trauma, surgery-induced metastatic disease, sepsis, weight loss, anorexia, fever control, cachexia, wound healing, ulcers, bowel barrier function, allergy, asthma, respiratory disorders, circulatory bios, coronary heart disease, anemia, blood clotting system disorders, kidney disease, central nervous system disorders, liver disease, ischemia, nutritional disorders, osteoporosis, endocrine disorders, epidermal disorders, psoriasis, acne vulgaris, panic disorder, behavioral disorder and / or post-traumatic stress disorder.
The bifidobacterial strain, as described here, acts by
i gonism and exclusion of pro-inflammatory microorganisms from the gastrointestinal tract. : The invention also provides a bifidobacterial strain or a - formulation, as described here, for use in the preparation of bio-therapeutic anti-inflammatory agents to reduce the levels of pro-inflammatory cytokines.
The bifidobacterial strain, as described here, can be used as a probiotic anti-infective strain.
The invention also provides a bifidobacterial strain or a formulation, as described here, for use in the prophylaxis and / or treatment of bipolar disorder, depression, mood disorders and / or anxiety disorders. BR The invention also provides a bifidobacterial strain or a formulation, as described here, that can be used as a cognitive enhancer for prophylaxis and / or for the treatment of central nervous system disorders, such as Alzheimer's disease, schizophrenia and / or disorder mild cognitive.
The invention also provides a bifidobacterial strain or a formulation, as described here, for use in the prophylaxis and / or treatment of obesity-related inflammation.
The invention also provides a bifidobacterial strain or a formulation, as described here, for use in the prophylaxis and / or treatment of obesity-related metabolic dysregulation.
We describe the strain of bifidobacteria AH1714 (NCIMB 41676) or mutants or variants thereof. The mutant can be a genetically modified mutant. The variant may be a variation in the natural occurrence of bifidobacteria. There is also a description of a rifampin-resistant variant of strain AH1714. The strain may be a probiotic. It may be in the form of a biologically pure culture.
We also describe an isolated NCIMB 41676 bifidobacterial strain. Bifidobacterial strains may be in the form of viable cells. Alternatively, the strains of bifidobacteria are in the form of non-viable cells. The general use of probiotic bacteria is under the form of. viable cells. However, it can also be extended to non-viable cells such as dead cultures or compositions containing beneficial factors - expressed by the probiotic bacteria. This may include thermally killed microorganisms or microorganisms killed by exposure to altered pH or exposure to pressure or gamma irradiation. With non-viable cells, product preparation is simplified, cells can be easily incorporated into drugs and storage requirements are much less limited than with viable cells. Lactobacillus casei YIT 9018 offers an example of the effectiveness of using heat-killed cells as a method of treating and / or preventing tumor growth, as described in US patent No. 4347240.. Bifidobacterial strains can be isolated from the colonic biopsy tissue of healthy human individuals, with bifido-bacterial strains being significantly immunomodulatory following oral consumption in humans.
We also describe a formulation that comprises the bifidobacterial strain as described here. The formulation can include other probiotic material. The formulation can include a prebiotic material. Preferably, the formulation includes an ingestible vehicle. The ingestible vehicle can be a pharmaceutically acceptable vehicle such as a capsule, tablet or powder. Preferably, the ingestible vehicle is a food product such as acidified milk, yogurt, frozen yogurt, powdered milk, milk concentrate, curd, sauces or drinks. The formulation can further comprise a protein and / or peptide, in particular proteins and / or peptides that are rich in glutamine / glutamate, a lipid, a carbohydrate, a vitamin, mineral and / or microelement. The bifidobacterial strain can be present in the formulation in more than 10º cfu per gram of the delivery system. Preferably, the formulation includes one or more of an adjuvant, a bacterial component, a drug entity or a biological compound.
We also describe a bifidobacterial strain or a formulation for use as food products, as a medicine, for use in prophylaxis and / or in the treatment of undesirable inflammatory activity, - for use in the treatment of undesirable respiratory inflammatory activity such as asthma, for use in prophylaxis and / or in the treatment of undesirable gastrointestinal inflammatory activity such as inflammatory bowel disease, for example: Crohn's disease or ulcerative colitis, irritable bowel syndrome, buritis or post-infectious colitis, for use in prophylaxis and / or treatment of gastrointestinal cancer, for use in prophylaxis and / or treatment of systemic disease such as rheumatoid arthritis, for use in prophylaxis and / or in the treatment of autoimmune disorders due to undesirable inflammatory activity, for use in prophylaxis and / or cancer treatment due to undesirable inflammatory activity, for use in cancer prophylaxis, for use in the prophylaxis and / or treatment of disease d iarreica due to undesirable inflammatory activity, co-. m Clostridium difficile-associated diarrhea, rotavirus-associated diarrhea or post-infective diarrhea, for use in the prophylaxis and / or treatment of diarrheal disease due to an infectious agent, such as E. coli.
We also describe a bifidobacterial strain or a formulation of the invention for use in the preparation of an anti-inflammatory biotherapeutic agent for prophylaxis and / or for the treatment of unwanted inflammatory activity or for use in the preparation of anti-inflammatory biotherapeutic agents for prophylaxis and / or treatment of undesirable inflammatory activity. The strain can act by antagonizing and excluding pro-inflammatory microorganisms from the gastrointestinal tract.
We also describe a strain of bifidobacteria or a formulation for use in the preparation of anti-inflammatory biotherapeutic agents to reduce levels of pro-inflammatory cytokines.
The bifidobacterial strain can be used in the preparation of anti-inflammatory biotherapeutic agents to modify the levels of 11-10.
The bifidobacterial strain can be used as an anti-infective probiotic due to its ability to antagonize the growth of species and pathogens.
We found that particular strains of bifidobacteria obtain immunomodulatory effects in vitro.
The invention is therefore of great potential therapeutic value in - prophylaxis or treatment of deregulated immune responses, such as unwanted inflammatory reactions, for example, asthma. - Bifidobacteria are commensal microorganisms. They have been isolated from microbial flora within the human gastrointestinal tract. The immune system in the gastrointestinal tract cannot have a pronounced reaction in relation to the members of this flora, since the resulting inflammatory activity would also destroy the host cells and the function of the tissue. Therefore, there are some mechanisms for the immune system to recognize non-pathogenic commensal members of the gastrointestinal flora as being different from pathogenic organisms. This ensures that lesions to the host tissue are restricted and that a defensive barrier. still be maintained. A deposit of Bifidobacterium longum AH1714 strain was made in the National Collections of Industrial and Marine Bacteria Limited (NCIMB) Ferguson Building, Craibstone Estate, Bucksburn region, Aberdeen, AB21 9YA, Scotland, UK on 5 November 2009 and read the NCIMB 41676 registration number.
Bifidobacterium longum can be a genetically modified mutant or it can be a naturally occurring variant of it.
Preferably, Bifidobacterium longum can be presented as viable cells.
Alternatively, Bifidobacterium longum may be in the form of non-viable cells.
It will be understood that the invention-specific bifidobacterial strain can be administered to animals (including humans) in an orally ingestible form in a conventional preparation such as capsules, microcapsules, tablets, granules, powder, lozenges, pills, suppositories, pensions and syrups. Suitable formulations can be prepared by commonly used methods using conventional organic and inorganic additives. The amount of active ingredient in the medical composition can be at a level that will exert the desired therapeutic effect.
The formulation can also include a bacterial component,. a pharmacological entity or a biological compound. In addition, a vaccine comprising the strains of the invention - can be prepared using any suitable known method and can include a pharmaceutically acceptable carrier or adjuvant.
Throughout the specification, the terms mutant, variant and genetically modified mutant include a strain of bifidobacteria whose genetic and / or phenotypic properties are altered in comparison to the parent strain. Naturally occurring variants of Bifidobacterium fongum include spontaneous changes in selectively isolated target properties. The deliberate alteration of the properties of the progenitor strain is achieved by conventional (in vitro) genetic manipulation technologies, such as genetic disturbance, conjugative transfer, etc. Genetic modification includes the introduction of exogenous and / or endogenous DNA sequences into the genome of a bifidobacterial strain, for example by insertion into the genome of the bacterial strain by vectors, including plasmid or bacteriophage DNA.
Natural or induced mutations include at least changes in a single base such as deletion, insertion, transversion or other DNA modifications that may result in alteration of the amino acid sequence encoded by the DNA sequence.
The terms mutant, variant and genetically modified mutant also include a strain of bifidobacteria that has undergone genetic changes that accumulate in a genome at a rate that is consistent in nature with all microorganisms and / or genetic changes that occur through spontaneous mutation and / or gene capture and / or gene loss that is not achieved by deliberate (in vitro) manipulation of the genome, but is achieved through the natural selection of variants and / or mutants that provide a selective advantage to support the survival of the bacteria when exposed to environmental pressures, such as antibiotics. A mutant can be created by the deliberate insertion (in vitro) of specific genes into the genome that do not fundamentally alter biochemical functionality.
organism, but whose products can be used to identify it. bacteria selection or selection, eg antibiotic resistance. i A person skilled in the art would recognize that mutant or variant bifidobacterial strains can be identified by analyzing DNA sequence homology with the parent strain. Strains of bifidobacteria that have a sequence identity close to that of the parent strain are considered mutant or variant strains. A strain of bifidobacterium with a sequence identity (homology) of 968% or more, such as 97% or more, or 98% or more, or 99% or more with the DNA sequence of the parent can be considered a mutant or variant. The sequence homology can be determined using the algorithm of the online program "BLAST" (an algorithm for searching for homology), publicly available at http: /Mwww.ncbi.nIlm.nih.gov / BLAST /. Mutants of the parent strain also include strains of bifido-bacteria having at least 85% sequence homology, such as at least 90% sequence homology, or at least 95% sequence homology for the intergenic spacer polynucleotide sequence from 16s - 23s of the parent strain. These mutants may further comprise DNA mutations in other DNA sequences in the bacterial genome.
BRIEF DESCRIPTION OF THE DRAWINGS The invention will be more clearly understood through the following description provided by way of example only, with reference to the attached drawings, in which: Figure 1 is a graph illustrating the transit of B. longum AH1714 through the gastrointestinal tract. Figure 2 is a photo of B. longum AH1714 grown on a Congo red agar plate; Figure 3 is a bar graph illustrating the I11L-10: 1L-12p70 ratio of peripheral blood mononuclear cells (PBMC) stimulated with the strain Bifidobacterium longum 1714 (bifidobacterium 1714); Figure 4 is a bar graph showing the induction profile of I1L-10 in splenocytes isolated from mice administered with 1714
'and with FTS, with and without in vivo challenge of lipopolysaccharide LPS of 1 mg / kg. In vitro cells are unstimulated (A), or are stimulated with lipopolysaccharide LPS (B) or stimulated with anticCD3 / CD28 (C). The data - are shown as the average &EPM; Figure 5 is a bar graph showing the TNF-a induction profile in splenocytes isolated from mice administered with 1714 and with FTS, with and without in vivo challenge of lipopolysaccharide LPS in vivo of 1 mg / kg. In vitro cells are either unstimulated (A) or stimulated with antiCD3 / CD28 (B). The data is shown as the average &EPM; Figure 6 is a bar graph showing the IFN-y induction profile in isolated splenocytes from mice administered with. 1714 and with FTS, with and without in vivo challenge of lipopolysaccharide LPS of 1 mg / kg. In vitro cells are either unstimulated (A) or stimulated with anti-D3 / CD28 (B). The data is shown as the mean & EPM.
Figure 7 is a bar graph showing the induction profile of I11L-12p70 in splenocytes isolated from mice administered with 1714 and with FTS, with and without challenge in vivo with 1 mg / kg lipopolysaccharide (LPS). In vitro cells are unstimulated (A) or stimulated with anti-CD3 / CD28 (B). The data is shown as the average &EPM; Figure 8 is a bar graph showing the induction profile of TNF-a (A) and 11-10 (B) in serum samples from mice administered with 1714 and with FTS 2 h after challenge in vivo with lipopolysaccharide (LPS) of 1 mg / kg. The data is shown as the average &EPM; Figure 9 is a bar graph showing the NFKB activity (photons / second) of the spleen isolated 3 hours after the challenge with a single dose of 0.5 mg / kg lipopolysaccharide (LPS), from animals fed with placebo € 1714 (* * indicates p <0.01); Figure 10 is a bar graph (A) showing the activity —NFKkB (photons / second) of images of the total body 1 h30 m after the challenge with a single dose of 0.5 mg / kg of lipopolysaccharide LPS, from animals fed with placebo and with 1714 ((B) and (C) are representative images
of the total body in black and white and in color; . Figure 11 is a bar graph representing the mobility time displayed by the mouse during a 6-minute test; - Figure 12 is a line graph representing the percentage of freezing in response to the feared stimulus (context) for day 1 (capture), day 2 (memory / extinction) and day 3 (extinction); Figure 13 is a line graph representing the percentage of freezing in response to the feared stimulus (clue) for day 1 (capture), day 2 (memory / extinction) and day 3 (extinction); Figure 14 is a bar graph representing the number of spheres hidden by the mouse during a 30-minute session; i Figure 15 is a bar graph representing the variation of. body temperature (TA) that the mice exhibited after treatment; and Figure 16 is a bar graph showing changes in cytokine levels in stimulated splenocytes from the rat model induced by diet.
DETAILED DESCRIPTION OF THE INVENTION A deposit of Bifidobacterium longum AH1714 strain was made in the National Collections of Industrial and Marine Bacteria Limited (NCIMB) Ferguson Building, Craibstone Estate, Bucksburn region, Aberdeen city, AB21 9YA, Scotland, United Kingdom on November 5, 2009 and read the NCIMB registration number 41676. A deposit of Bifidobacterium longum strain UCC35624 was made at the National Collections of Industrial and Marine Bacteria Limited (NCIMB) Ferguson Building, Craibstone Estate, Bucksbum region, city of Aberdeen, AB21 9YA, Scotland, United Kingdom on January 13, 1999 and took the NCIMB registration number 41003. Examples The following examples describe and demonstrate in more detail the modalities within the scope of the present invention. The examples are provided for illustrative purposes only and should not be considered as limitations to the present invention, since many variations of it are possible, without deviating from the character and scope of the invention. Example 1 - Isolation of Bifidobacterium longum AH1714. The strain of Bifidobacterium longum AH1714 was isolated from the colonic biopsy tissue of healthy human subjects.
Sections of the thick TGI, obtained during colorectal probing, were analyzed for probiotic bacterial strains. Mucous tissue from the human gastrointestinal tract was transferred to a collection tube containing phosphate buffered saline (PBS), supplemented with 0.05 cysteine-HCl. Triton X-100 (0.05%) was added to release adherent microorganisms from the tissue sample. Tissue samples were then B incubated for 10 min. The samples were shaken vigorously and lactobacilli and bifidobacteria were isolated from gastrointestinal tissue by plates on selective agar (De Man, Rogosa and Sharpe agar (MRS) + vancomycin and Wilkins-Chalgren + mupirocin agar, respectively). Isolated colonies were then removed from the plates and reapplied by smearing three times to ensure purity. Evaluations by microscopic examination, Gram stains, catalase tests, fructose-6-phosphate phosphoquetolase were used to determine likely bifidobacterial species and the isolates were placed in a buffer solution with 40% glycerol and stored at -20 oe - BO0ºC. Sequencing of the intergenic spacer 168 region was used to confirm the identity of the newly isolated strains.
Following the isolation of a pure and post-bifidobacterial strain assigned the designation AH1714, the microbiological characteristics were assessed and are summarized in Table 1 below. AH1714 is a gram-positive, pleomorphic, catalase-negative bacterium, which is fructose-6-phosphate phosphoquetolase positive, confirming its identity as a bifidobacterium.
Table 1 - Physico-chemical characteristics of B. / ongum AH1714 | [LL eae | [mare == 168-23s intergenic spacer (IGS) sequencing was performed to identify the isolated bifidobacterial species.
Briefly, DNA was isolated from AH1714 using 100 µl of solution for extraction and 25 µl solution for tissue preparation (Sigma, XNAT2 kit). The samples were incubated for 5 minutes at room temperature followed by 2 hours at 95ºC and then 100 µl of neutralization solution (Sigma, XNAT2 kit) were added.
The genomic DNA solution was quantified using a spectrophotometer and stored at 4ºC.
PCR (polymerase chain reaction) was performed using the IGS primer (intergenic spacer). The primer pairs used were IGS R S-CTGGTGCCAAGGCATCCA-3 '(SEQ | D No. 4) and IGS L 5- GCTGGATCACCTCCTTTCT-3' (SEQ ID No. 3). The cycle conditions were 94ºC for 4 minutes (1 cycle), 94ºC for 45 seconds, 53ºC for 45 seconds, 72ºC for 45 seconds (28 cycles). The PCR reaction contained 2 μl (100 ng) of DNA, PCR mixture (Sigma, Red Taq), 0.025 nM of IGS L and R primer (MWG Biotech, Germany). PCR reactions were performed on a Biotherma thermocycle.
The PCR products (10 µl) were passed, together with a molecular weight marker (100 bp Ladder, Rock) through a 2% agarose gel stained with ethidium bromide (EtBr) in TAE (tris-acetate EDTA), to determine your IGS profile.
The bifidobacterium PCR products (single band) were purified using the Promega Wizard PCR purification kit.
The purified PCR products were sequenced using primers (above) for the intergenic - spacer region (IGS). The sequence data was then searched against the NCB! I nucleotide database to determine the strain identity by nucleotide homology.
The resulting DNA sequence data was submitted to the BLAST search tool for men.
NCBI standard nucleotide-to-nucleotide methodology - (http: // www. ncbi.nlm.nih.govwBLASTY /). The closest match for the string was identified and then the strings were aligned for - comparison using the DNASTAR MegAlign software.
The sequences (SEQ ID No. 1 [IGS straight sequence] and SEQ ID No. 2 [IGS reverse sequence]) obtained can be seen in the sequence listing.
Searching the NCIMB database revealed that AH1714 has a unique IGS sequence (SEQ ID No. 1 [forward sequence] and SEQ ID No. 2 [reverse sequence]) with its sequence homology closer to a Bifidobacterium longum .
In order to be able to develop a barcode PCR profile for AH1714, PCR was performed using BOX primers (8). The cycle conditions were 94ºC for 7 min. (1 cycle); 94ºC for 1 minute,. 53ºC for 45 seconds, 65ºC for 8 minutes, (30 cycles) and 65ºC for 16 minutes.
The PCR reaction contained 50 ng of DNA, PCR mixture (Sigma, Red Taq) and 0.038 nM of BOXAIR primer (5 CTACGGCAAGGC- GACGCTGACG-3 ') (SEQ ID No. 5) (MWG Biotech, Germany). PCR reactions were performed on a Biotherma thermocycle.
The PCR products were passed through a 3% agarose gel together with a molecular weight marker (Roche, 100 bp ladder) and photographed.
Antibiotic sensitivity profiles.
The antibiotic sensitivity profiles for B. longum AH1714 were determined using the 'susceptibility disk' assay. Cultures were grown in the appropriate broth medium for 48 h and spread (100 μl) across the plate on the agar medium.
Discs containing known concentrations of antibiotics were placed on the agar.
The strains were examined for antibiotic sensitivity after 1 to 2 days of incubation at 37ºC under anaerobic conditions.
Table 2 - Antibiotic resistance [Girl | Antiiotic Flatama Mo
| | Genmamena - | Aminagicaside antibiotic [mM | | Acidonaidic | Synthetic qumoloma armbiômico | Ro | | sutametorazole | Amphbiotic sufonamide É Ro | suramanaia | | "Sulfametoxazol metapra IQ [Cetera [O Ts - Shit | Antibioticonitoimidazo - | mM [| Mmupraena OO RA 'R = Resistant (Zone size <14 mm) M = Moderately sensitive (Zone size 15 to 19 mm) S = Sensitive (Zone size 2 20 mm) Intestinal transit To determine whether Bifidobacterium longum AH1714 can survive at low pH values, equivalent to those found in the stomach, bacterial cells were harvested from fresh cultures overnight. , washed twice in phosphate buffer (pH 6.5) and resuspended in TPY broth adjusted to pH 2.5 (with 1 M HCI) The cells were incubated at 37ºC and survival was measured at intervals of 5, 30, 60 and 120 minutes using the plate counting method.
AH1714 survived well for 5 minutes at pH 2.5 while no viable cells were recovered after 30 minutes.
Upon leaving the stomach, putative probiotics are exposed to bile salts in the small intestine.
In order to determine the ability of B. longum AH1714 to survive exposure to bile, cultures were applied by smearing on TPY agar plates supplemented with 0.3% (weight by volume), 0.5%, 1 %, 2%, 5%, 7.5% or 10% of pork bile.
The growth of B. longum AH1714 was observed in the plates that contained up to 0.5% bile.
i Table 3 - Growth of AH1714 in the presence of pork bile (result of duplicates): [ema [00 [os os 1020 [so 75 [100] [arizia | [ar [Da +++ = very good growth - 100% ++ = good growth -66% + = unsatisfactory growth -33% - = no growth-0% In a germ-free murine model, B. fon's ability - AH1714 gum to move in the gastrointestinal tract has been evaluated.
The mice consumed 1x10 0º AH1714 daily and the presence of 210 fecal pellets of the ingested microorganism was examined.
The detection of AH1714 was facilitated by isolating a spontaneous rifampin-resistant variant of bifidobacterium - the incorporation of rifampicin in the RCA + cysteine plates used to assess movement (transit) ensures that only the rifampin-resistant bifidobacterium was grown .
Fecal samples were collected daily and the transit of B. longum AH1714 through the gastrointestinal tract was confirmed (see Figure 1) Antimicrobial activity To assess the antimicrobial activities of B. longum AH1714 in relation to indicator cultures and to determine whether the antimicrobial activity was due to acid production, AH1714 was grown overnight in MRS (supplemented with 0.05% cysteine-HCI). 2 ul of AH1714 culture was noted on the agar and incubated for 24 h.
Indicator organisms were grown in TSB (E. coli and Salmonella typhimurium), Brucella broth (Campylobacter jejuni) and Clostridium reinforced medium (SmPC, Clostridium difficile). The indicator grass was prepared by inoculating a melted layer with 2% (v / v) of the indicator culture overnight, which was then poured onto the surface of the painted probiotic cultures, followed by overnight growth. on the agar plates.
The plates were incubated at 37ºC under conditions suitable for the individual strain.
and the recorded growth after 24-48 h.
Opening zones greater than 1 mm in diameter were considered sensitive to the probiotic strain.
This test was also carried out in a medium supplemented with 2% B-glycerophosphate. and as a buffering agent to limit antagonistic activity due to acid production.
Table 4 - Antimicrobial activity of AH1714 o voadembiçãoimm | sampylobacterigamt | the QU | Cstidiumperííngens | 20 [0 | Generation of rifampicin (Rif ”) resistant strains of 1714 'In order to be able to track the movement (transit) of AH1714 in faecal samples, a spontaneous rifampicin-resistant variant (rif +) was isolated as follows: a fresh broth culture of AH1714 was spread over the plate (100 ul) over MRS + rifampicin + cysteine with the lowest concentration of rifampicin (in the range of 0.1%, 0.08%, 0.06%, 0.04%, 0.02% € 0.002%). Plaque medium without rifampicin was included as a positive control.
Both sets of plates were incubated anaerobically at 37ºC (48 hours). The purity of the removed plates was then assessed before choosing a colony on the agar plate supplemented with rifampicin and applied by smear on the nearby rifampicin supplemented plate of higher concentration.
In addition, a colony was removed by smearing the MRS agar plate, applied to a new MRS agar plate and both sets of plates incubated anaerobically at 37ºC (48 hours). This process was repeated for the entire range of plaques supplemented with famicin.
A single colony of a fully mature culture on an MRS agar plate supplemented with 50 pg / mL rifampicin was used to inoculate the 20 ml MRS broth and the resulting culture used for subsequent storage.
The variant's identity was confirmed by microscopic evaluation, IGS sequence analysis and specific PCR analysis.
Example 2 - Congo red agar screen
A congo red agar screen was used to evaluate notably strains of bacteria that express extracellular polysaccharide (EPS - extracellular polysaccharide). A 10 ml Rogosa broth medium - modified (+ 0.05% cysteine) was briefly inoculated aseptically with a newly cultivated colony of the bacterial strain and incubated anaerobically at 37ºC until turbidity (about 16 to 24 hours). The broth cultures were aseptically spread by smearing the Congo red agar plates and incubated anaerobically at 37ºC for 48 hours.
It is believed that the extracellular polysaccharide (EPS) produced as a subpro- —duct of growth and / or metabolism of certain strains prevents the absorption of Congo red dye, resulting in a colony morphology with a cream / white color.
Dyes that produce less EPS absorb: easily the Congo red dye, resulting in a pink / red colony morphology.
Strains that do not produce a red EPS color appear almost transparent on a red agar background.
Referring to Figure 2, the colony morphology of B. longum AH1714 is of convex, mucoid and bright white colonies.
Example 3 - Bifidobacterium 1714 induces a significantly elevated 11-10: 11-12 ratio.
Peripheral blood mononuclear cells (PBMCs) were isolated from healthy peripheral human blood using CPT tubes, BD Vacutainer (BD catalog 362761), according to the manufacturer's instructions.
PBMCs were washed and resuspended in Dulbecco-Glutamax '"modified Eagle's medium (Glutamax (glutamine substitute) + pyruvate + 4.5 g / l glucose (Gibco catalog 10569-010), 10% fetal bovine serum ( catalog Sigma F4135) and 1% penicillin / streptomycin (catalog Sigma PO781) The PBMCs were incubated (2 x 10 th cells per well) in flat-bottom plates with 96 wells and 20 ul of a bacterial suspension (in a concentration of 10x10 'ufeuml) were added.
PBMCs were matched with bacteria for 48 hours at 37ºC / 5% CO, in an incubator.
After the 2-day incubation period, the plates were centrifuged at 300 x g and the supernatants were removed and stored frozen at -80ºC until analysis. The levels of interleukin-10 (IL-10) and interleukin-12p70 (IL-12p70) in the supernatant cultures were quantified using a kit. 96-well test kit available from Meso Scale Discovery (Gaithersburg, MD, USA; catalog K15008B-1). The bacteria were prepared for co-culture experiments in two formats: (a) Freshly grown bacteria were grown in Difco MRS medium and harvested shortly after entering the stationary phase, All cells were grown under anaerobic conditions at 37ºC. (b) Bacteria were grown under anaerobic conditions at 37ºC in Difco MRS medium and harvested shortly after entering the stationary phase. Freeze-dried powders were generated for each of these bacteria and stored at -80ºC in 100 mg pre-aliquot flasks. Immediately before use, an aliquot of each strain was removed from the freezer and left outdoors until it reached room temperature. Each strain was washed 3 times in 10 ml of Ringer's solution followed by centrifugation. A new bottle was used on each occasion. Growth curves (DO vs. number of living cells) were created for each growth condition and the washed cells were normalized by the number of cells prior to addition to PBMCs. A control on bacteria was also included in all experiments. All tests were performed in triplicate. The results are shown in Figure 3.
The control of inflammatory diseases is exercised at several levels. Control factors include hormones, prostaglandins, reactive oxygen and nitrogen intermediates, leukotrienes and cytokines. Cytokines are biologically active proteins of low molecular weight that are involved in the generation and control of immune and inflammatory responses. Several cells produce these cytokines, with neutrophils, monocytes, and lymphocytes being the major sources during inflammatory reactions due to their large number in the injured occlusion. Multiple mechanisms exist, by which cytokines generated at inflammatory sites influence the inflammatory response. The chemotaxis
i xia stimulates the flow of inflammatory cells to the lesion site, while certain cytokines promote the infiltration of cells into tissues.
Cytokines released at the site of the injured tissue result in the activation of the in- infiltrate. flammable.
Most cytokines are pleiotropic and express multiple biologically overlapping activities.
Since uncontrolled inflammatory responses can result in diseases, such as IBS, it is reasonable to imagine that cytokine production has taken an uncertain turn in individuals affected by these diseases.
Interleukin-10 (IL-10) is an anti-inflammatory cytokine that is produced by many types of cells, including monocytes, macrophages, dendritic cells, mast cells and lymphocytes (in particular regulatory cells: T). 11-10 downregulates the expression of pro-Th1i cytokines. inflammatory, MHC class antigens |! and costimulatory molecules in cells presenting antigens.
It also improves cell survival, proliferation and antibody production.
This cytokine can block NF-xB activity and is involved in the regulation of the JAK-STAT signaling pathway.
Knockout studies in mice have shown the essential role of 11-10 in immunoregulation when mice with IL-10KO developed severe colitis.
In addition, bacteria that are potent inducers of 11-10 have been shown to be able to promote regulatory T cell differentiation in vivo, thus contributing to immune homeostasis (7; 8). Interleukin-12 (11-12) is a pro-inflammatory cytokine associated with the polarization of T cell responses with Th1 effector and stimulates the production of other pro-inflammatory Th1 cytokines, such as interferon-gamma (IFN-y) and tumor necrosis factor-alpha (TNF-a), of T cells and natural killers (NK). High levels of expression 11-12 are associated with autoimmunity.
The administration of IL-12 to people suffering from autoimmune diseases worsened the symptoms of the disease.
In contrast, mice with 1L-12 knockout or treatment of mice with antibodies - neutralizers of | l-12 alleviated the disease.
Cytokine cascades and networks control the inflammatory response, rather than the action of a particular cytokine on a particular cell type. The relative levels of expression or balance of two cytokines (such as 1L-10 and IL-12) are more informative than the expression of a single cytokine. In these studies, we stimulate human PBMCs with ”a range of different bacterial strains. All strains induced I1L-10 and all of the strains induced IL-12. However, examination of the ratio of IL-10 to IL-12 induction revealed that some strains of bacteria induced a higher ratio (example, more I1L-10 with less | L-12) compared to other strains. This is an important observation, since it is the balance between each of these opposing signals that ultimately determines the immunological result.
It is expected that a high ratio of IL-10: 1L-12 will promote an anti-inflammatory response associated with adequate immunoregulatory activity while a low ratio of [1L-10: 11-12 will contribute to the Th1i polarization of the immune response. Thus, the CMSP IL-10: 11-12 ratio is an important selection criterion for identifying bacterial strains with immunoregulatory properties.
Example 4 - The prolonged feeding of mice with Bif. AH1714 is associated with an increase in anti-inflammatory cytokine IL-10 and a decrease in Th1 and pro-inflammatory cytokines TNF-a IFN-y and 11-12 in healthy animals and in a sepsis / inflammation model.
Materials & Methods: Female mice aged 6 to 8 weeks are purchased from Harlan UK (United Kingdom) and housed in individually ventilated cages with ad libitum access to food and sterile water for mice.
Mice of similar weight are randomly assigned to 2 groups and administered FTS (control vehicle n = 9), Bifidobacterium longum strain AH1714 (n = 17) via esophageal tube daily for 115 days, Following the administration period, blood is collected from 10 AH1714 mice and 6 control vehicles are challenged with 1 mg / kg delipopolysaccharide LPS (Sigma, L4391) via intraperitoneal injection.
Following the administration period, blood is collected from 6 AH1714 mice and 4 control vehicles, the serum is extracted and preserved
i vado for measuring cytokine.
The spleen is also removed and single cell suspensions are cultured in vitro. Cytokines are measured in supernatant cells following 48 hours of culture. . In addition, 10 more AH1714 mice and 6 control mice are administered with a single dose of lipopolysaccharide LPS at 1 mg / kg via intraperitoneal injection.
After 2 hours the blood is collected and the mice slaughtered.
Serum and splenocyte cells were treated and analyzed as previously described.
Splenocyte cytokine assay Splenocytes are isolated from the spleen and incubated for 48 hours at 37ºC (in the presence of penicillin and streptomycin) with control medium, lipopolysaccharide LPS or anticD3 / CD28. Cytokines in supernatants. cultures are tested using a 96-well assay kit available from Meso Scale Discovery (Gaithersbura, MD, USA; catalog K15008B-1). Interleukin 1 beta (Il-1b), interleukin 6 (I-6), interleukin 8 (1-8) interleukin 10 (11-10), interleukin 12p70 (1112p70), Interferon-gamma (IFN-y) and alpha tumor necrosis (TNFa) are quantified and reported as picograms per milliliter (pg / mL). Cytokine serum assay Serum is analyzed using IL-10 mouse from Meso Scale Discovery and the ultra-sensitive TNF-a kit.
Results The long-term feeding of mice (115 days) with Bif.
AH1714 is associated with an increase in ex vivo anti-inflammatory cytokine I1-10 of stimulated ex vivo CMSPs, compared with the placebo group (fed with FTS) for healthy mice (see Figure 4 (B)) or in a sepsis / inflammation model (mouse challenged with LPS; see Figure 4 (C)). Long-term feeding of mice (115 days) with Bif AH1I714 is associated with a decrease in the Th1 and pro-inflammatory cytokines TNF-a IFN-y and IL-12 (sub unit p70) of stimulated ex vivo CMSPs, compared with the placebo group (fed with FTS) in a sepsis / inflammation model (mouse challenged with LPS; see Figure 5 (B), Figure 6 (B) and Figure 7 (B)). Long - term feeding of mice (115 days) with. Bif.
AH1714 is associated with an increase in serum levels of anti-inflammatory cytokine 11-10 and a decrease in Thi and pro-inflammatory cytokines TNF-a compared to the placebo group (fed with FTS) in a sepsis / inflammation model (mouse challenged with LPS see Figure 8 (A & B)). Taken together, these results demonstrate that the 1714 strain of Bifidobacterium longum has in-vivo systemic immunomodulatory activity and anti-inflammatory activity and protects against LPS or TLR-A4-mediated inflammatory responses. - Example 5 - Bif 1714 has immunomodulatory activity when combined with cells of the human immune system, different from Bif.
AH35624. Materials & Methods The Bifidobacterium longum infant strain UCC35624 (B624), two independent culture lots (1 & 2) and the Bifidobacterium longgum 1714 strain are tested using a cytokine induction assay in PBMC.
The bacteria are prepared for co-culture experiments in the following formats.
The bacteria are grown under anaerobic conditions at 37ºC in Difco MRS medium and harvested shortly after entering the stationary phase.
Freeze-dried powders are generated for each of these bacteria and stored at -80ºC in 100 mg pre-aliquot flasks.
Immediately before use, an aliquot of each strain is removed from the freezer and left outdoors until it reaches room temperature. Each strain is washed 3 times in 10 ml of Ringer's solution followed by centrifugation.
A new bottle is used on each occasion.
Direct microscopic counts are performed using a Petroff-Hausser counting chamber, according to the manufacturer's instructions, and the washed cells are normalized by the number of cells prior to the PBMC assay.
The bacteria (20 µl in phosphate buffered saline (PBS - phosphate buffered saline)) are added
in each PBMC well to result in the total number of bacteria, as indicated for each experiment.
Cytokine induction assay in PBMC (peripheral blood mononuclear cells)
Peripheral blood mononuclear cells (PBMC) are isolated from healthy human peripheral blood using BD Vacutainer CPT tubes (cell catalog 362761), according to the manufacturer's instructions.
PBMCs are washed and resuspended in Eagle's medium modified by Dulbecco - Glutamax TM (Glutamax (substituted
to glutamine) + pyruvate + 4.5 g / L glucose (Gibco catalog 10569-010), 10% fetal bovine serum (Sigma catalog F4135), and 1% penicillin / streptomycin (Sigma catalog PO781). PBMCs are incubated (2 x 10 th cells per well.) In flat bottom plates with 96 wells and 20 µl. of a bacterial suspension.
Control is also carried out without bacteria.
All tests are performed in triplicate.
After a 2-day incubation at 37 ° C, the plates were shaken at 300 x q and the supernatants were removed and stored frozen at -80 ° C until analysis.
PBMC are matched with the bacteria for 48 hours at 37ºC / 5% CO in an incubator.
After the 2-day incubation period, the plates are centrifuged at 300 x 9, and the supernatants removed and stored frozen at -80ºC until analysis.
Cytokines in culture supernatants are tested using a 96-well assay kit available from Meso Scale Discovery (Gaithersburg, MD, USA; catalog K15008B-1). Human interleukin 1 beet (11-1b), human interleukin 86 (II-6), human interleukin 8 (11-8) human interleukin 10 (II-10), human interleukin 12p70 (I112p70), Interferon-gamma human (IFN-y) and tumor necrosis factor alpha (TNFa) are quantified and reported as picograms per milliliter (pg / mL). Each sample is tested in duplicate. Results The strain of Bifidobacterium longum infantil UCC35624 (B624), two independent culture lots (1 & 2) and the strain of Bifidobacterium longum 1714 are tested for immunomodulation using an
i cytokine production in PBMC with 1.0E + 07 bacteria.
The oO supernatants are tested for a range of cytokines including IL-18, -6, -8, -10 and -12, TNF-a and IFN-y. . Compared to 35624 (both cultures that showed a similar pattern for all tested cytokines), strain 1714 exhibited a very similar pattern for many of the tested cytokines.
Surprisingly, however, 1714 presented a very different pattern for I1L-12, IFNy and IL-6. 11-6: Incubation with 1714 induces a significantly lower level of IL-6 compared to 35624 in 1.0 x 10 ”bacteria per well (see Table 5) Table 11-12: Incubation with 1714 induces a significantly lower level of IL-12 compared to 35624 in 1.0 x 10 th bacteria per well (see Table 6) Incubation of INF-y with 1714 induces a significantly lower level of INF-y compared to 35624 in 1.0 x 10 th bacteria per well (see Table 6) Table 6 bacteria) (pg / ml) (-pgiml) (pg / ml) (pg / ml) [these | 500 | so so iso [me | 2 Te TB a Foligne et al ” demonstrated that strains of lactic acid-forming bacteria exhibiting an ability to induce higher levels of anti-inflammatory cytokine I1L-10 and lower levels of inflammatory cytokine 11-12 offered the best protection in the in vivo colitis model whereas, in contrast , strains leading to a low ratio of cytokine IL-10 / I1L-12 could not significantly alleviate the symptoms of colitis.
The in vivo protection observed was strain specific. The cytokine profile obtained for Bif. AH1714 could suggest that this strain has the potential for enhanced efficacy in the in vivo ulcerative colitis model. : IL-6 is a cytokine strongly linked in the pathology of DIL. L-6 is relevant to many disease processes such as diabetes, atherosclerosis, depression, Alzheimer's disease, systemic lupus erythematosus and rheumatoid arthritis. Therefore, there is an interest in developing anti-IL-6 agents as a therapy against many of these diseases. Example 6 - A Bif. AH1714 reduces LPS-induced NFKB activity in a murine sepsis / inflammation model in vivo Materials & Methods NFKkBlux transgenic mice in a background - CS7BL / 6J-CBA / J are obtained from Charles River Laboratories (Wilmington, USA) and bred in-house . The mice are housed under maintained barrier conditions.
Females are administered Bif. AH1714, as a dry and frozen powder reconstituted in water in approximately 1x10º colony-forming units, either per day, or per animal, or a place control. Mice consume microorganisms in their water ad libitum for 20 days prior to the challenge with LPS.
NFKB activity is measured after administration of the luciferin substrate and depicted using the Xenogen IVIS 100. The baseline NFKB activity is measured prior to the challenge with a single dose of 0.5 mb / kg of LPS. After 3 hours, all animals are then portrayed. The NFKB activity of the total body is assessed by subtracting the baseline readings.
All animals are then slaughtered and the spleen, liver, small intestine and colon are removed and placed in a culture dish for individual images.
Results A Bif. AH1714 reduces LPS-induced systemic NFKB activity in a murine sepsis / inflammation model in-vivo, as demonstrated
i drawn by a decrease in NFKB activity in isolated spleens 3 hours a- CS post challenge (see Figure 9) and images of the total animal 1 h 30 m after challenge (see Figure 10) from animals fed 1714 compared to . animals fed with placebo.
These results demonstrate that feeding with Bif. 1714 is associated with a reduced level of systemic inflammation associated with the transcription factor NFKB.
Example 7 - 1714 exhibits positive benefits in animal models of depression and anxiety, Depression and anxiety are the most common psychiatric disorders with a high prevalence rate in the community.
Anxiety disorders are generally subdivided into panic disorder, generalized anxiety disorder, post-traumatic disorder and obsessional disorder. compulsive, modern antidepressants, such as selective serotonin uptake inhibitors (eg, fluoxetine) and selective noradrenergic and serotonin uptake inhibitors (eg, venlafaxine) are widely used to treat these disorders.
However, treatments are not always effective and are not acceptable to patients.
There is a need to develop alternative strategies.
The possibility that —probiotics may be effective in such conditions is suggested by the previous data indicating that the probiotic Bifidobacterium Infantis reduces the corticosterone stress hormone in rodents (9). Here we examine the behavioral effects of Bifidobacteri- an AH1714 in stress models in mice and compare it to a widely used SSRI, specifically escitalopram, which is used to treat both anxiety and depression.
The animals are treated with escitalopram, or AH1714 for three weeks.
Material & Methods Tail suspension test A well-characterized test to assess depression activity or anti-lepression activity.
The mice are individually suspended by the tail on an annular bar on a horizontal support (dis-
ground level = 30 cm) using tape (distance from tail tip = 0 cm). Typically, mice demonstrate various escape-oriented behaviors interspersed with a temporary increase in the fight against immobility. A 6-minute test session is employed and recorded on video. The videos are subsequently scored by a highly trained observer who is unaware of the treatment. The recorded parameter is the number of seconds spent immobilized.
Fear conditioning test Widely used to assess the cognitive components of anxiety disorders. We use a three-day protocol that allows for: observation of contextual and track-related fear learning. After 3 minutes of exploring their contextual environment, the mice receive - 6 pairs of 20 seconds of a specific track (10 KHz tone, 70dB combined with turning on the device's light), combined at the end with a light shock ( 0.4 mA) of 2 seconds, followed by 1 minute of exposure to the context only. The procedure is repeated in the following two days, however, without shock and the freezing behavior to the context or clue is observed. The first day allows assessing the ability of the intervention to change the strength of the context and the conditioning of the method induced by clues, while the third day allows the observation of the extinction of fear learning. Extinction is the formation of new memories, and drugs that facilitate extinction can play a role! in the treatment of post-traumatic stress disorder.
The test of hiding spheres.
Proposed as a model of obsessive-compulsive disorder. Animals that are more anxious need to engage in active behavior (hide the spheres defensively) to avoid anxiogenic stimulation in the light-dark box and elevated labyrinths. The mice are placed individually in small cages, in which 20 spheres were equally distributed on the 5 cm deep sawdust surface, and a wire cover is placed on top of the cage. Mice are left undisturbed for 30 min and then hidden spheres are counted (for example, those that are more than and less than three quarters covered by sawdust). Results - In the tail suspension test, AH1714 gives a positive result suggesting possible antidepressant activity. With reference to Figure 11, AH1714 induced a shorter immobility time than the vehicle (Veh) which suggests attenuated depression behaviors in animals fed with 1714. This is similar to the impact of conventional antidepressants, such as Lexapro € O. In terms of cognition, in the fear conditioning test, test animals treated with AH1714 showed a positive learning effect. With reference to Figure 12, in the context of fear conditioning tests (mainly hippocampal and amygdala-dependent memories), 1714 induced a greater freeze to the context (Cxt) than the vehicle (Veh) on day 1 and day 2, with the same freezing percentage as the vehicle on day 3, this suggests that 1714 promotes learning of contextual fear and memory without harming extinction, suggesting a positive role in the contextual memory of fear events. With reference to Figure 13, in fear conditioning tests by clues (dependent on the amygdala), 1714 induced a greater freezing of the fear lane (stimulus) than the vehicle (Veh) on day 1, with the same freezing percentage as the vehicle on days 2 and 3. This suggests that 1714 promoted learning of fear dependent on the amygdala (clue) and memory, without harming memory and extinction, suggesting a positive role in the memory of stimuli of fear regardless of the context.
Evidence of a positive effect on obsessive-compulsive disorder arises from studies using the test to hide the spheres. Animals treated with AH1714 hid less spheres in the task of hiding spheres, which is indicative of less anxiety in animals fed with 1714 and suggests a possible effect on obsessive-compulsive disorder (Figure 14). As in the case of escitalopram, AH1714 induced a lower increase in body temperature caused by the stress of being handled
(decreased stress-induced hypothermia), this suggests a lower anxiety in animals fed with 1714 (Figure 24). There are no differences between the results with any intervention. - Conclusion AH1714 in animal models of depression and anxiety behaves similarly to conventional antidepressants.
The observed impact is similar to that reported in the literature for antidepressants such as SSIRs (selective serotonin uptake inhibitors). As a whole, the data indicate that AH1714 may be beneficial in the treatment of psychiatric syndromes of depression and anxiety.
Example 8: 1714 exhibits positive benefits in inflammatory markers in diet-induced obesity. . In recent years, it has been well established that obesity is associated with low-grade inflammation that contributes to the development of pathologies associated with obesity, which include type 2 diabetes mellitus (T2D), cardiovascular disease (CVD), hypertension , hypercholesterolemia, hypertriglyceridemia and non-alcoholic fatty liver disease.
Visceral fat produces a number of inflammatory cytokines and chemokines (such as leptin, tumor necrosis factor-a (TNF-a), chemo-attractive macrophage protein-1 and interleukin-6, among others), in which the production it can be pathologically deregulated in the state of obesity (analyzed by Shoelson et al., 2007). In fact, while macrophages are thought to contribute significantly to insulin resistance, other studies have suggested that controlling the anti-inflammatory properties of cells with a potentially regulatory phenotype may have therapeutic potential.
A recent study suggests that Tre cells reduce the inflammatory state of adipose tissue and, thus, insulin resistance in mice (Fuerzer et al., 2009). In addition, a seminal body of work implied abnormalities in the gastrointestinal tract microbiota as a driving force for metabolic deregulation related to obesity, suggesting that interventions aimed at the health of the gastrointestinal tract will have beneficial health effects in metabolic disorders related to obesity.
It has been suggested that the gastrointestinal tract microbiota may be involved in the development of obesity in the regulation of energy homeostasis, in insulin resistance, non-alcoholic fatty liver diseases and in the metabolism of energy, lipid and amino acid (analyzed by Ley ef a /., 2009) Diet-induced obesity (DIO) in the mouse model was chosen as the most appropriate mouse model to assess the impact of selected probiotic candidates on obesity and metabolic health and for observe the relationship between obesity and inflammatory marks. The DIO mouse model refers to healthy mice fed on a high-fat diet to induce obesity over time.
| Experimental development - Seven week old male C57BL / J6 mice were fed a low fat diet (10% of calories from fat; Re-search Diets, New Jersey, USA;% D12450B), a high fat diet (DIO ; 45% of calories from fat; Research Diets, New Jersey, USA; fD12451) or a high fat diet with AH1714 (1 x 10º cfu / day) in drinking water for 14 weeks. All mice were housed in groups of 5 and aliquots of fresh probiotics were administered daily. Body weight and food consumption were assessed weekly. At the end of 14 weeks, the mice were sacrificed and the internal organs were removed, weighed and stored at -80ºC. The spleens were removed and splenocyte cytokine assays were performed as in Example 4. Results As expected, DIO mice gained significantly more fat mass (p <0.001) compared to light controls during the 14 weeks of the feeding period. In agreement with previous studies, DIO mice consumed significantly more calories than light controls, as measured by cumulative caloric intake during the 14-week study period (p <0.001). In splenocytes stimulated by LPS (stimulation of innate immunity) of DIO mice, AH1714 had the effect of reducing TNFa and cytoplasmic response.
i 11 to 12 LPS (Figure 16). In splenocytes stimulated by CD3 / CD28 CO (adaptive immunity stimulus), treatment with AH1714 had the effect of reducing the cytokine IL6 response. These results indicate a "systemic anti-inflammatory effect in the DIO mouse model consistent with CSMS data and in vivo mouse model data illustrated in other examples.
Immunomodulation The human immune system plays a significant role in the etiology and pathology of a wide variety of human diseases. Hyper- and hypo-immune responses result in, or are a component of, most: sick states. A family of biological entities, called cytokines, is particularly important in controlling the immunological process. Disorders of these delicate cytokine networks are increasingly associated with many diseases. These diseases include, but are not limited to, inflammatory disorders, immunodeficiency, inflammatory bowel disease, irritable bowel syndrome, cancer (particularly those of the gastrointestinal and immune systems), diarrheal disease, diarrhea associated with the use of antibiotics, pediatric diarrhea, appendicitis, autoimmune disorders, multiple sclerosis, Alzheimer's disease, rheumatoid arthritis, celiac disease, diabetes mellitus, organ transplant, bacterial infections, viral infections, fungal infections, periodontal disease, urogenital disease, disease sexually transmitted, HIV infection, HIV replication, HIV-associated diarrhea, surgery-associated trauma, surgery-induced metastatic disease, sepsis, weight loss, anorexia, fever control, cachexia, wound healing , ulcers, bowel barrier function, allergy, asthma, respiratory disorders, circulatory disorders, coronary heart disease, anemia, coagulatory system disorders blood action, kidney disease, central nervous system disorders, liver disease, ischemia, nutritional disorders, osteoporosis, endocrine disorders, epidermal disorders, psoriasis and acne vulgaris The effects on cytokine production are specific for each of the probiotic strains examined. Thus, probiotic strains are
can be selected to normalize an exclusive imbalance, CO in particular of cytokine, for a specific type of disease.
Customization of disease-specific therapies can be achieved using "a single AH1714 strain or mutants and variants thereof or a selection of these strains.
Immune education Enteric flora is important for the development and proper function of the intestinal immune system.
In the absence of an enteric flora, the intestinal immune system is underdeveloped, as demonstrated in the models of germ-free animals, and certain functional parameters are reduced, such as macrophage phagocytic ability and immunoglobulin production (10). The importance of the flora of the gastrointestinal tract in stimulating responses. non-harmful immune systems is becoming more evident.
The increase in the incidence and severity of allergies in the western world has been linked to an increase in hygiene and sanitization, concomitant with a decrease in the number and scope of infectious challenges encountered by the host.
This lack of immune stimulation may allow the host to react to non-pathogenic but antigenic agents, resulting in an allergy or autoimmunity.
The deliberate consumption of a series of non-pathogenic immunomodulatory bacteria will provide the host with the appropriate and necessary educational stimulus for the correct development and control of immune function.
Inflammation Inflammation is the term used to describe the accumulation of local fluid, plasma proteins and white blood cells in a location that has sustained physical injury, infection or where there is an immune response in progress.
The control of the inflammatory response is exercised at a number of levels (11). Control factors include cytokines, hormones (eg hydrocortisone), prostaglandins, reactive intermediates and leukotrienes.
Cytokines are biologically active proteins of low molecular weight that are involved in the generation and control of immune and inflammatory responses, while also regulating development, tissue repair and hemato-
poiese.
They provide a means of communication between leukocytes and also with other types of cells.
Most cytokines are pleiotropic and express multiple biologically overlapping activities.
Cascades and "cytokine networks control the inflammatory response rather than the action of a particular cytokine on a particular cell type (12). The weakening of the inflammatory response results in low concentrations of the appropriate activation signals and other inflammatory mediators. leading to the cessation of the inflammatory response.
TNFa is a pivotal pro-inflammatory cytokine because it initiates a cascade of cytokines and biological effects resulting in the inflammatory state.
Therefore, agents that inhibit TNFα are currently being used for the treatment of inflammatory diseases, e.g. infliximab. . Pro-inflammatory cytokines are thought to play a large role in the pathogenesis of many inflammatory diseases, including inflammatory bowel disease (IBD). Current therapies to treat ID are aimed at reducing the levels of these pro-inflammatory cytokines, including IL-8 and TNFa.
Such therapies can also play a significant role in the treatment of systemic inflammatory diseases such as rheumatoid arthritis.
The strains of the present invention may have potential for application in the treatment of a range of inflammatory diseases, particularly if used in combination with other anti-inflammatory therapies, such as non-steroidal anti-inflammatory drugs (NSAIDs) or Infliximab.
Cytokines and cancer The production of multifunctional cytokines by a wide range of tumor types suggests that significant inflammatory responses are underway in cancer patients.
It is currently uncertain what protective effect this response has in relation to the growth and development of tumor cells in vivo.
However, these inflammatory responses can adversely affect the tumor patient.
Complex cytokine interactions are involved in regulating cytokine production and cell proliferation within the tumor and normal tissues (13, 14). It has long been recognized that weight loss (cachexia) is the only cause
i the most common death in cancer patients and the initial malnutrition O indicates a poor prognosis. For a tumor to grow and spread, it must induce the formation of new blood vessels and degrade the extracellular matrix. home. The inflammatory response can play a significant role in the above mechanisms, thus contributing to host decline and tumor progression. Due to the anti-inflammatory properties of childhood Bifidobacterium longum, these bacterial strains can reduce the rate of malignant cell transformation. In addition, intestinal bacteria can produce substances with genotoxic, carcinogenic and tumor-promoting activity from dietary compounds and bacteria from the gastrointestinal tract can activate the reactive pro-carcinogenic agents for DNA (15). In general, Bifidobacterium species have low activities of xenobiotic metabolizing enzymes compared to other populations within the gastrointestinal tract such as bacteroids, eubacteria and clostridia. Therefore, increasing the number of Bifidobacterium bacteria in the gastrointestinal tract can beneficially modify the levels of these enzymes.
Vaccine / medication application Most pathogenic organisms gain entry via mucous surfaces. Effective vaccination of these sites protects against invasion by a particular infectious agent. Oral vaccination strategies have been concentrated, until today, on the use of live attenuated pathogenic organisms or purified encapsulated antigens (16). Probiotic bacteria, developed to produce antigens of an infectious agent, in vivo, can provide an attractive alternative because these bacteria are considered safe for human consumption (GRAS state).
Studies in mice have shown that consumption of probiotic bacteria that express foreign antigens can elicit protective immune responses. The gene that encodes the C toxin fragment (TTFC - tetanus toxin fragment OC) was expressed in Lacfococceus lactis and the mice were immunized orally. The system was able to induce antibodies at significantly high concentrations to protect mice from a lethal toxin challenge. In addition to the presentation of
tigen, vectors of live bacteria can produce bioactive compounds, CO as immunostimulatory cytokines, in vivo.
The secretion of bioactive human IL-2 or IL-6 and TTFC by L. factis induced serum IgG concentrations - 10 to 15 times higher in intranasally immunized mice (17). However, with this particular bacterial strain, the total IgA level was not increased by coexpression with these cytokines.
Other bacterial strains, such as Streptococcus gordonii, are also being examined for their usefulness as mucosal vaccines.
The colonization of the vaginal and oral cavities of murine by recombinant S. gordonii induced both responses of mucosal and systemic antibodies to the antigens expressed by this bacterium (18). Thus, oral immunization using probiotic bacteria as vectors would not only protect the host from infection. tion, but it can also replace the immune stimulus that the pathogen would normally use, thus contributing to the host's immunological education.
Prebiotics The introduction of probiotic organisms is carried out by ingesting the microorganisms in a suitable vehicle.
It would be advantageous to provide a means that could promote the growth of these probiotic strains in the large intestine.
The addition of one or more oligosaccharides, polysaccharides or other prebiotics accentuates the growth of lactic acid-forming bacteria in the gastrointestinal tract.
The term prebiotic refers to any non-viable food component that is specifically fermented in the colon by indigenous bacteria that are thought to be of positive value, eg, bifidobacteria, lactobacillus.
Types of prebiotics can include those that contain fructose, xylose, soy, galactose, glucose and mannose.
The combined administration of a probiotic strain with one or more prebiotic compounds can improve the growth of the probiotic administered in vivo, resulting in a more pronounced health benefit, and is called a symbiotic.
Other active ingredients It will be understood that probiotic strains can be administered prophylactically or as a method of treatment or alone,
or with other probiotic and / or prebiotic materials, as described above. "In addition, the bacteria can be used as part of a prophylactic or treatment regimen using other active materials, such as those used to treat inflammation or other disorders, especially those with an immune involvement. Such combinations can be administered in a simple formulation or in separate formulations administered at the same time or at different times and using the same or different routes of administration.
The dimensions and values presented in the present invention should not be understood as being strictly limited to the exact: numerical values mentioned. Instead, except where otherwise specified, each of these dimensions is meant to mean both value. mentioned as a range of functionally equivalent values around that value. For example, a dimension displayed as "40 mm" is intended to mean "about 40 mm".
All documents cited in the detailed description of the Invention are, for the most part, incorporated herein by reference, and the citation of any document should not be construed as admitting that it represents prior art with respect to the present invention. If - there is a conflict between any meaning or definition of a term mentioned in this document and the meaning or definition of the same term in a document incorporated by reference, the meaning or definition attributed to the term mentioned in this document will take precedence.
Although particular embodiments of the present invention have been illustrated and described, it should be apparent to those skilled in the art that various other changes and modifications can be made without departing from the character and scope of the invention. Therefore, it is intended to cover in the appended claims all such changes and modifications that fall within the scope of the present invention.
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BUDAPEST TREATY ON RECOGNITION. INTERNATIONAL DEPOSIT OF MICRO-ORGANISMS FOR. EFFECTS OF PATENT PROCEDURES Alimentary Health Ltd Building 2800 DEPOSIT RECEIPT. Cork Airport Business Park Kinaale Road INTERNATIONAL IN THE EVENT OF AN ORIGINAL DEPOSIT Cork awarded according to Rule 7.1 by the INTERNATIONAL DEPOSITORY AUTHORITY identified at the end of this page NAME AND ADDRESS OF
DEPOSITANTE LL IDENTIFICATION OF MICRO-CRGANISM Identification reference given by the Access number given by. DEPOSITOR AUTHORITY DEPOSIT INTERNATIONAL Bifidobacterium fongums AHI714 NCIMB 41676
1. - SCIENTIFIC DESCRIPTION AND / OR PROPOSED TAXONOMIC DESIGNATION The microorganism identified under the item | above was accompanied by: o a scientific description a proposed taxonomic designation (please tick with a cross where applicable) Ul. - RECEIPT AND ACCEPTANCE This international depositary authority accepts the micro-organism identified in item | above, which was received by the same on November 5, 2009 (date of the original deposit) "Nv. - RECEIPT OF REQUEST FOR CONVERSION The microorganism identified in item | above was received by this international depositary authority on (date of original deposit) and a request to convert the original deposit into a deposit under the Budapest Treaty was received by her on (date of receipt of the request for conversion) V. - INTERNATIONAL DEPOSITORY AUTHORITY Name: - NCIMS Ltd Signature (s) of the person (s) (s) with the power to represent F Boi international depositary authority or authorized erguson Building (s): Craibstone Esnic furtnia Der Address: Buciksbum Date: November 11, 2009 Aberdeen 'ABZ19YA 1; Where Rule 6 / 4 (d) if applicable, such date is the date on which the state of the international depositary authority was received Form BP / 4 (all page)

权利要求:
Claims (38)
[1]
CLAIMS - 1. Isolated strain of NCIMB 41676 bifidobacterium.
[2]
2. Bifidobacterial strain according to claim 1, which is in the form of viable cells.
[3]
3. Bifidobacterial strain according to claim 1, which is in the form of non-viable cells.
[4]
4, Bifidobacterium strain, according to any one of claims 1 to 3, in which the bifidobacterium is isolated from the colon biopsy tissue of a healthy human being.
[5]
5. Bifidobacterial strain, according to any of the kings. vindications | to 4, which is significantly immunomodulatory after con. oral juice. '
[6]
A formulation comprising a bifidobacterial strain as defined in any one of claims 1 to 5.
[7]
A formulation according to claim 6, which further comprises a probiotic material.
[8]
A formulation according to claim 6 or 7, which further comprises a probiotic material.
[9]
A formulation according to any one of claims 6a8, further comprising an ingestible vehicle.
[10]
A formulation according to claim 9, in which the ingestible vehicle is a pharmaceutically acceptable carrier, such as a capsule, tablet or powder.
[11]
11. Formulation according to claim 9, in which the carrier is a food product such as acidified milk, yogurt, frozen yogurt, powdered milk, concentrated milk, curd, sauces or drinks.
[12]
12. Formulation according to any one of claims 6 to 11, which further comprises a protein and / or a peptide, in particular proteins and / or peptides which are rich in glutamine - glutamate, a lipid , a carbohydrate, a vitamin, a mineral and / or a microelement.
[13]
13. Formulation according to any of the claims
sections 8 to 12, in which the bifidobacterial strain is present in an amount of more than 10º cfu per gram of the formulation.
[14]
A formulation according to any one of claims 6 to 13, which further comprises an adjuvant.
[15]
A formulation according to any one of claims 6 to 14, which further comprises a bacterial component.
[16]
16. Formulation according to any one of claims 6 to 15, which further comprises a drug entity.
[17]
17. Formulation according to any one of claims 6 to 6, which further comprises a biological compound. .:
[18]
18. Formulation according to any one of claims 6 to 17, which is used for immunization and vaccination protocols. '
[19]
19. Bifidobacterial strain according to any one of claims 7 to 5, or formulation according to any of claims 6 to 18 for use in food products.
[20]
20. Bifidobacterial strain according to any one of claims 1 to 5, or formulation according to any of claims 6 to 18, for use as a medicine.
[21]
21. Bifidobacterial strain according to any one of claims 1 to 5, or formulation according to any one of claims 6 to 18, for use in prophylaxis and / or in the treatment of unwanted inflammatory activity.
[22]
22. Bifidobacterial strain, according to any one of claims 1 to 5 or formulation, according to any of claims 6 to 18, for use in prophylaxis and / or in the treatment of unwanted gastrointestinal inflammatory activity, such as disease inflammatory bowel, for example: Crohn's disease or ulcerative colitis, irritable bowel syndrome; bursitis; or post-infectious colitis.
[23]
23. Bifidobacterial strain according to any of claims 1 to 5, or formulation according to any of claims 6 to 18, for use in the prophylaxis and / or treatment of gastrointestinal cancer.
[24]
24. Bifidobacterial strain, according to any of the. claims 1 to 5 or formulation according to any one of claims 6 to 18, for use in prophylaxis and / or in the treatment of systemic diseases such as rheumatoid arthritis.
[25]
25. Bifidobacterial strain according to any one of claims 1 to 5 or formulation according to any one of claims 6 to 18, for use in prophylaxis and / or in the treatment of autoimmune disorders due to inflammatory activity unwanted.
[26]
26. Bifidobacterial strain according to any one of claims 1 to 5, or formulation according to any one of claims 6 to 18, for use in the prophylaxis and / or treatment of cancer due to unwanted inflammatory activity. ,
[27]
27. Bifidobacterial strain according to any one of claims 1 to 5, or formulation according to any of claims 6 to 18, for use in cancer prophylaxis.
[28]
28. Bifidobacterial strain according to any of claims 1 to 5, or formulation according to any of claims 6 to 18, for use in prophylaxis and / or in the treatment of diarrheal diseases due to unwanted inflammatory activity, such as diarrhea associated with Clostridium difficile, diarrhea associated with rotavirus, or post-infectious diarrhea or diarrheal disease caused by an infectious agent such as E. coli.
[29]
29. Bifidobacterial strain according to any one of claims 1 to 5 or formulation according to any one of claims 6 to 18 for use in the preparation of anti-inflammatory biotherapeutic agents for prophylaxis and / or treatment of unwanted inflammatory activity.
[30]
30. Bifidobacterial strains according to claim 29 for use in the preparation of a panel of biotherapeutic agents to modify IL-10 levels.
[31]
31. Bifidobacterial strain according to any one of claims 1 to 5 or formulation according to any one of claims 6 to 18, for use in the prevention and / or treatment of inflammatory disorders
diseases, immunodeficiency, inflammatory bowel disease, bowel syndrome. irritable bowel, cancer (particularly of the gastrointestinal and immune systems), diarrheal disease, antibiotic-associated diarrhea, pediatric diarrhea, appendicitis, autoimmune disorders, multiple sclerosis, Alzheimer's disease, rheumatoid arthritis, celiac disease, diabetes mellitus, organ transplantation, bacterial infections, viral infections, fungal infections, periodontal disease, urogenital disease, sexually transmitted disease, HIV infection, HIV replication, HIV-associated diarrhea, trauma associated with surgery, induced metastatic disease by surgery, sepsis, weight loss, anorexia, fever control, cachexia, wound healing, ulcers, bar- bowel disease, allergy, asthma, respiratory disorders, circulatory disorders, coronary heart disease, anemia, blood clotting system disorders, kidney disease, central nervous system disorders, liver disease, ischemia, nutritional disorders , osteoporosis, endocrine disorders, epidermal disorders, psoriasis, acne vulgaris, panic disorder, behavioral disorder and / or post-traumatic stress disorder.
[32]
32. Bifidobacterial strain according to any one of claims 1 to 5, wherein the strain acts by antagonizing and excluding pro-inflammatory microorganisms from the gastrointestinal tract.
[33]
33. Bifidobacterial strain according to any one of claims 1 to 5 or formulation according to any one of claims 6 to 18 for use in the preparation of anti-inflammatory biotherapeutic agents for the reduction of pro-inflammatory cytokine levels .
[34]
34. Use of a bifidobacterial strain, as defined in any one of claims 1 to 5, as a probiotic anti-infectious strain.
[35]
35. Bifidobacterial strain according to any one of claims 1 to 5 or formulation according to any one of claims 6 to 18, for use in the prophylaxis and / or treatment of bipolar disorder, depression, mood and / or anxiety disorders.
[36]
36. Use of a bifidobacterial strain, as defined in any one of claims 1 to 5 or the formulation, as defined in any of claims 6 to 18, as a congnitive enhancer for the prophylaxis and / or treatment of disorders of the central nervous system, like. Alzheimer's disease, schizophrenia and / or mild cognitive impairment.
[37]
37. Use of a bifidobacterial strain as defined in any of claims 1 to 5 or the formulation as defined in - any of claims 6 to 18 for the prophylaxis and / or treatment of inflammation related to obesity.
[38]
38. Use of a bifidobacterial strain as defined in any one of claims 1 to 5 or the formulation as defined in any of claims 6 to 18 for the prophylaxis and / or treatment of obesity-related metabolic dysregulation .

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PL3745872T3|2018-01-29|2022-01-31|Precisionbiotics Group Limited|A combination product for prophylaxis and treatment of irritable bowel syndrome|

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US20200375204A1|2018-01-29|2020-12-03|Precisionbiotics Group Limited|Bifidobacterium longum ncimb 41676|

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EP3746098A1|2018-01-29|2020-12-09|PrecisionBiotics Group Limited|Bifidobacterium longum|

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WO2019145570A1|2018-01-29|2019-08-01|Alimentary Health Limited|A combination product for prophylaxis and treatment of irritable bowel syndrome|

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CN111918663A|2018-01-29|2020-11-10|精密生物集团有限公司|Bifidobacterium longum NCIMB 41676|

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CN110964656B|2018-09-30|2021-05-28|内蒙古伊利实业集团股份有限公司|Bifidobacterium lactis BL-99 capable of preventing osteoporosis and application thereof|

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CN109929773B|2019-01-10|2020-06-19|江苏德禧生物科技有限公司|Bifidobacterium capable of being used for selenium-rich culture and active protein and application thereof|

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CN110974917A|2019-11-25|2020-04-10|垒途智能教科技术研究院江苏有限公司|Compound preparation with auxiliary effect of preventing and treating psychological depression of children and teenagers|

法律状态:
2020-09-08| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2020-09-08| B15I| Others concerning applications: loss of priority|Free format text: PERDA DA PRIORIDADE REQUERIDA US 61/344,030 DE 11.05.2010, POIS POSSUI DEPOSITANTE DIFERENTE DO INFORMADO NA ENTRADA NA FASE NACIONAL E SUA RESPECTIVA CESSAO NAO FOI APRESENTADA, MOTIVO PELO QUAL SERA DADA PERDA DESTA PRIORIDADE, CONFORME AS DISPOSICOES PREVISTAS NA LEI 9.279 DE 14/05/1996 (LPI) ART. 167O. |

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2020-11-10| B25A| Requested transfer of rights approved|Owner name: ALIMENTARY HEALTH LIMITED (IE) |

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2020-11-17| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2020-12-29| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-01-19| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-07-06| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-10-13| B25D| Requested change of name of applicant approved|Owner name: PRECISIONBIOTICS GROUP LIMITED (IE) |

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2021-11-23| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2021-11-30| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2022-01-25| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US12/616,752|US9771624B2|2008-11-11|2009-11-11|Bifidobacterium longum|

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US12/616,752|2009-11-11|

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US34403010P| true| 2010-05-11|2010-05-11|

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US61/344,030|2010-05-11|

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PCT/IE2010/000066|WO2011058535A1|2009-11-11|2010-11-11|Probiotic bifidobacterium strain|

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