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    • 专利摘要:
    The present invention relates to a method of lyophilizing a faecal microbiota sample from a donor subject, comprising the steps of: A) mixing a faecal microbiota sample of a donor subject with a diluent selected from the group consisting of polyols, di- pentasaccharides, maltodextrins and mixtures thereof, and B) freezing the mixture obtained in A) at a temperature below -50 ° C, preferably between -70 ° C and -100 ° C, and its lyophilization.
    公开号:FR3045383A1
    申请号:FR1562750
    申请日:2015-12-18
    公开日:2017-06-23
    发明作者:Herve Affagard；Carole Schwintner；Catherine Juste；Joel Dore；Audrey Chapron；Fernanda Fonseca；Olivier David
    申请人:Institut National de la Recherche Agronomique INRA；Maat Pharma SA；
    IPC主号:
    专利说明:

    Method for lyophilizing a fecal microbiota sample
    The present invention relates to a method of lyophilizing a fecal microbiota sample. The invention also relates to the use of the lyophilizate obtained in the transplantation of fecal microbiota, preferably for treating intestinal dysbiosis, particularly Clostridium difficile infections.
    The human intestinal microbiota is the set of micro-organisms (bacteria, yeasts and fungi) found in the human gastrointestinal system (stomach, intestine and colon). Microbial diversity is currently estimated at about 10 bacterial species making up the dominant intestinal microbiota of an adult, with an abundance of 1014 bacteria, representing a dominant bacterial metagenome of 200,000 to 800,000 genes in each individual, ie 10 to 50 times the number of genes in the human genome.
    Sterile in utero, the intestine colonizes from the first days of life until evolving towards a single individual microbiota. Each person has relatively similar bacteria in terms of species, but the exact composition of its microbiota (species, proportions) is largely (more than% of species) specific to the host.
    Thus, the human intestinal microbiota is a very diverse ecosystem, complex and specific to each individual.
    It is essential for the health of an individual to maintain a stable microbiota that is both able to return to its original state after a change and resistant to invasion. Maintaining a great diversity of the microbiota promotes its stability. However, some pathologies or treatments unbalance the microbiota: antibiotics, for example, as well as diseases with an inflammatory component, such as inflammatory bowel disease (IBD), can limit the diversity of the microbiota in the intestine.
    Antibiotic treatments (or antibiotics), in particular, result in an alteration of the microbiota and a loss of its barrier functions, which can promote the proliferation of pathogenic organisms such as Clostridium difficile.
    Clostridium difficile infections are responsible for nosocomial diarrhea; this bacterium may have resistance to classical antibiotic therapy (broad spectrum, such as vancomycin or metronidazole). In order to restore the intestinal microbiota, and to fight against Clostridium difficile infections, and thus restore homeostasis (i.e. symbiosis), fecal microbiota transplantation is considered and tested. It consists of introducing the stool of a healthy donor into the gastrointestinal tract of a recipient patient to rebalance the altered intestinal microbiota of the host. This fecal microbiota transplant is generally allogeneic (that is, from a healthy donor individual to a patient). The results obtained on Clostridium difficile-type infections are encouraging, and some patients have been successfully treated (Tauxe et al, Lab Medicine, Winter 2015, Volume 46, Number 1 or Van Nood E, Speelman P, Nieuwdorp M, Keller J 2014 Fecal Microbiota Transplantation: Facts and Controversies Curr Opin Gastroenterol 30 (1): 34-9).
    However, the current transplantation method is empirical and takes no special precautions to best preserve the viability of anaerobic bacteria, major components of the gut microbiota. In addition, the efficiency of fecal microbiota transplantation is variable, and may require more than one cure.
    There is therefore a need for safe, efficient and easily obtainable fecal microbiota samples, especially on an industrial scale. In addition, there is a need for fecal microbiota samples in which the viability of the bacteria is conserved, and having a long shelf life.
    The present invention makes it possible to meet these needs.
    The present invention thus relates to a process for preparing a fecal microbiota lyophilizate of a donor subject, comprising the following steps: A) mixing a faecal microbiota sample of a donor subject with a diluent selected from polyols, di-pentasaccharides, maltodextrins and mixtures thereof, and B) freezing the mixture obtained in A) at a temperature below -50 ° C, preferably between -70 ° C and -100 ° C, then freeze-drying.
    Preferably, the fecal microbiota sample used in step A) is previously purified.
    Preferably, the present invention relates to a method for preparing a fecal microbiota lyophilizate of a donor subject, comprising the steps of:
    Al) optionally, the preparation of a continuous gradient of iodixanol or 5- (N-2,3-dihydroxypropylacetamido) -2,4,6-triiodo-N, N'-bis (2,3 dihydroxypropyl) isophthalamide formed by freeze-thawing, A2) mixing at least one faecal microbiota sample of a donor subject with a saline buffer optionally comprising iodixanol or 5- (N-2,3-dihydroxypropylacetamido) -2 , 4, 6-tri-iodo-N, N'-bis (2,3-dihydroxypropyl) isophthalamide, anaerobically, A3) optionally, the deposition of the mixture obtained in A2) under the gradient obtained in Al), A4) the centrifugation sequential low acceleration anaerobiosis, or ultracentrifugation of the mixture obtained in A2) or A3), A5) recovery of the bacterial ring or the supernatant formed at the end of step A4), under anaerobic conditions, A6) the mixture of the bacterial ring or the supernatant recovered in A5) with a diluent chosen from polyols, di- pentasaccharides, maltodextrins and mixtures thereof, and B) freezing the mixture obtained in A6) at a temperature below -50 ° C, preferably between -70 ° C and -100 ° C, and lyophilization.
    In such a method, step A1), as well as de facto step A3), are optional.
    Such a method is indeed easy to implement, and its effectiveness can be estimated by comparing the microbial population obtained after the process, compared to the initial sample.
    The present invention also relates to the use of a fecal microbiota lyophilizate obtainable by the process according to the invention, as a research tool in functional genomics, in metaproteomics or in immunology.
    For example, the lyophilizate obtainable by the process according to the invention can be used to generate bacterial pellets which, immobilized in agarose matrices (or other gels), allow the extraction of DNA fragments from very large size, used for cloning and functional studies. It can also be used to prepare cytosolic protein extracts or envelope proteins from bacterial communities for metaproteomic assays. Finally, it can be used to study the recognition of intact bacteria by the immune system of the host.
    The present invention also relates to the use of a fecal microbiota lyophilizate of a donor subject that can be obtained by the method according to the invention, in the transplantation of autologous or allogenic fecal microbiota.
    The present invention also relates to the use of a fecal microbiota lyophilizate of a donor subject that can be obtained by the process according to the invention, for treating intestinal dysbiosis, due to infections and especially Clostridium difficile, dysbiosis induced by medicinal treatments, by physical treatments (radiation in particular), by surgical interventions (intestinal in particular), by colonoscopies or by nutritional contributions. The present invention also relates to the use of a fecal microbiota lyophilizate of a donor subject that can be obtained by the method according to the invention, for treating a pathology chosen from inflammatory bowel diseases ( IBD), intestinal functional disorders, obesity, metabolic diseases (type 2 diabetes, metabolic syndrome in particular) and autoimmune diseases (especially type 1 diabetes), allergies, liver diseases (steatosis, cirrhosis in particular), certain neurological diseases (autism in particular) and certain cancers (colorectal cancer in particular). By intestinal dysbiosis, we mean any sustained imbalance of the gut microbiota. Sustained imbalance of the intestinal microbiota is understood to mean any loss of beneficial microorganisms, and / or any loss of microorganism diversity, and / or any expansion or development of aggressive microorganisms among commensals (pathobionts), and / or any proliferation of pathogenic micro-organisms (particularly C. difficile). Any sustained alteration of the human intestinal microbiota can indeed cause or accompany chronically a pathological state. In particular, the reduction of diversity within the microbiota is characteristic of diseases associated with dysbiosis (obesity, Crohn's disease, diabetes or allergy) (Sansonetti, Collège de France, January 22, 2014).
    Preferably, the pathology to be treated is intestinal dysbiosis.
    Inflammatory chronic diseases of the intestine (IBD) is understood to mean, in particular, Crohn's disease, ulcerative colitis.
    Functional bowel disorders include irritable bowel syndrome, spasmodic colitis.
    The method for preparing a fecal microbiota lyophilizate of a donor subject according to the invention thus comprises the following steps: A) mixing a sample of fecal microbiota of a donor subject with a diluent chosen from polyols, di-pentasaccharides, maltodextrins and mixtures thereof, and B) freezing the mixture obtained in A) at a temperature below -50 ° C., preferably between -70 ° C. and -100 ° C., and then lyophilizing it . Step A) of the process according to the invention comprises mixing a sample of fecal microbiota of a donor subject with a diluent selected from cryoprotectants such as polyols, di- pentasaccharides; bulking agents such as maltodextrins; and their mixtures.
    Preferably, the faecal microbiota sample of the donor subject is a stool sample of said donor. Indeed, the stool sample contains fecal microbiota of the donor subject. Preferably, according to the invention, the donor subject is a healthy human subject. By "healthy" subject, we mean a subject not suffering from an imbalance of the intestinal microbiota or a pathology diagnosed / recognized by the medical profession. Preferably, the fecal microbiota sample of the donor subject is first purified. Alternatively, preferably, according to the invention, the donor subject is a sick human subject.
    Preferably, the stool sample has a mass of at least 20 g. The fecal microbiota sample is still obtained and mixed anaerobically (i.e. under an oxygen free atmosphere). In anaerobiosis, the viability of the bacteria constituting the fecal microbiota present in the sample is thus preserved.
    Preferably, prior to its use, the fecal microbiota sample is filtered anaerobically. The prior filtration step may include, anaerobically, filtration using a Seward filter bag.
    Preferably, the fecal microbiota sample is removed anaerobically, using an airtight collection device. Preferably, this device is in a form of the type comprising: a container comprising a body which has an interior space adapted to receive the sample of fecal microbiota of the donor subject, and a neck which delimits an access opening to the interior space of the body, and a cover adapted to be removably and sealingly mounted on the neck of the container so as to close the access opening of the neck and to close the interior space of the body, in which the body the container consists of a flexible bag, and wherein at least one of the container and the lid is provided with a discharge member adapted to evacuate at least a portion of the gases contained in the interior space of the body of the container.
    Alternatively, the airtight collection device is in a form of the type comprising: a container comprising a body which comprises an interior space adapted to receive the sample of fecal microbiota of the donor subject, and a neck which delimits a access opening to the interior space of the body, and - a cover adapted to be removably and sealingly mounted on the neck of the container so as to close the access opening of the neck and to close the interior space of the body, wherein the interior space of the container body optionally comprises an oxygen neutralizing chemical device.
    The diluent may be chosen from the following compounds: cryoprotectants such as di- pentasaccharides, i.e. disaccharides, trisaccharides, quadrisaccharides and pentasaccharides; or polyols, such as glycerol, mannitol, sorbitol, propylene glycol or ethylene glycol, - fillers, such as partial hydrolysates of starch, in particular wheat or corn, or starch, comprising a large amount of maltodextrins, and mixtures thereof.
    Preferably, the diluent is an aqueous saline solution comprising at least one cryoprotectant and / or a filler. Thus, typically, the aqueous saline solution comprises water and physiologically acceptable salts. Typically, the salts are calcium, sodium, potassium or magnesium salts, with chloride, gluconate, acetate or hydrogencarbonate ions.
    The aqueous saline solution may also optionally comprise at least one antioxidant. The antioxidant is in particular chosen from ascorbic acid and its salts (ascorbate), tocopherols (especially Τα-tocopherol), cysteine and its salified forms (hydrochloride in particular) and their mixtures.
    Preferably, the aqueous saline solution comprises: at least one salt selected from sodium chloride, calcium chloride, magnesium chloride, potassium chloride, sodium gluconate and sodium acetate, and optionally at least one antioxidant, preferably selected from sodium L-ascorbate, tocopherols, L-cysteine hydrochloride monohydrate and mixtures thereof.
    Typically, the salt is present in the aqueous saline solution in a concentration of between 5 and 20 g / l, preferably between 7 and 10 g / l.
    Typically, the antioxidant is present in the aqueous saline solution in an amount of between 0.3 and 1% by weight relative to the total volume of solution, preferably in an amount of between 0.4 and 0.6% by weight relative to the total volume of solution.
    Preferably, when the antioxidant is a mixture of sodium L-ascorbate and L-cysteine hydrochloride monohydrate, the sodium L-ascorbate is present in an amount of between 0.4 and 0.6% by weight relative to the total volume of solution. , and the L-cysteine hydrochloride monohydrate is present in an amount of between 0.01 and 0.1% by weight relative to the total volume of solution.
    Preferably, the aqueous saline solution comprises at least one cryoprotectant. A cryoprotectant is a substance used to protect the sample from damage caused by freezing, particularly due to the formation of ice crystals.
    Preferably, the cryoprotectant is selected from polyols, di-pentasaccharides (disaccharides, trisaccharides, quadrisaccharides and pentasaccharides), and mixtures thereof. Preferably, the cryoprotectant is chosen from polyols, tri- and disaccharides and mixtures thereof. More preferably, the cryoprotectant present in the aqueous saline solution is a disaccharide or a trisaccharide.
    Among the polyols which may be used, glycerol, mannitol, sorbitol, but also propylene glycol or ethylene glycol may be found.
    Among the di-pentasaccharides that may be used, mention may be made of dimers, trimers, quadrimers and pentamers of identical or different units, said units being chosen from glucose, fructose, galactose, fucose and N-acetylneuraminic acid.
    Among the disaccharides that can be used are, in particular, trehalose or one of its analogues, or sucrose.
    These cryoprotectants can be used alone or as a mixture.
    Typically, the total amount of cryoprotectant present in the aqueous saline solution is between 3 and 30% by weight relative to the total volume of solution, preferably between 4% and 20% by weight relative to the total volume of solution.
    Preferably, the cryoprotectant is chosen from glycerol, mannitol, sorbitol, propylene glycol, ethylene glycol, trehalose and its analogues, sucrose, galactose-lactose and mixtures thereof. More preferably, the cryoprotectant is galactose-lactose or trehalose.
    Preferably, the aqueous saline solution according to the invention comprises at least one filler. The bulking agent is preferably selected from partial hydrolysates of starch or starch. Partial hydrolysates of starch, in particular wheat or maize, as well as partial hydrolysates of starch, for example of potato, comprise a large quantity of maltodextrins. Maltodextrins are the result of the partial hydrolysis of starch or starch, and consist of various sugars (glucose, maltose, maltotriose, oligo- and polysaccharides), the proportions of which vary according to the degree of hydrolysis.
    Preferably, the bulking agent present in the aqueous saline solution is a mixture of maltodextrins, wherein the amount of maltodextrins is between 4 and 20% by weight relative to the total volume of solution.
    Preferably, step A) is carried out by mixing the sample of fecal microbiota with the diluent, in a weight ratio of microbiota, preferably purified (g) / volume of diluent (mL) of between 1: 1 and 1: 10.
    Then the mixture obtained in A) is frozen at a temperature below -50 ° C, preferably between -70 ° C and -100 ° C, and then lyophilized: this is step B). It is preferably carried out under the following conditions:
    Bl) freezing the mixture obtained in A) at a temperature below -50 ° C, preferably between -70 ° C and -100 ° C, preferably at a temperature of about -80 ° C, B2) the loading the frozen mixture obtained in Bl) into a pre-cooled lyophilizer at a temperature between -50 ° C and -30 ° C, at atmospheric pressure, then B3) at least one primary drying step of the mixture loaded B2) comprising the lowering the pressure to a value between 80 and 200 μ bar (preferentially between 100 and 150 pbar) and then increasing the temperature of the shelves to a value between -20 ° C and + 25 ° C ( preferably -10 ° C.) by applying a heating rate of between 0.2 and 0.5 ° C./min. The pressure and temperature values of the shelves are chosen so that the temperature of the product is kept below the collapse temperature throughout the sublimation. The parameters are kept constant until the ice is completely removed from the mixture, then B4) the secondary drying of the mixture obtained in B3) comprising the lowering of the pressure to a value of less than or equal to 80 μ bar, preferably the minimum possible value for the equipment, and the elevation of the temperature of the shelves to a value between + 25 ° C and + 35 ° C, preferably 25 ° C at a heating rate of between 0.1 and 0, 3 ° C / min, and keep it there for 8 to 15 hours.
    Preferably, the freezing of step B1) is carried out at a temperature below -50 ° C, preferably between -70 ° C and -100 ° C. Preferably, the freezing temperature is between -70 ° C and -100 ° C; more preferably it is about -80 ° C or about -100 ° C. In order to be frozen, the mixture obtained in A) can be aliquoted beforehand, to ensure specimens of constant volume. The frozen sample is thus loaded into a pre-cooled lyophilizer at a temperature between -50 ° C and -30 ° C at atmospheric pressure; this is step B2).
    Then, the primary desiccation of the mixture charged B2) is involved; this is step B3). It comprises at least one primary drying step of the mixture charged with B2) comprising lowering the pressure to a value of between 80 and 200 pbar (preferably between 100 and 150 pbar) and then increasing the temperature of the shelves up to a value between -20 ° C and +25 ° C (preferably -10 ° C) by applying a heating rate of between 0.2 and 0.5 ° C / min. Shelf pressure and temperature values are chosen so that the temperature of the product is kept below the collapse temperature throughout the sublimation. The parameters are kept constant until the ice is completely removed from the mixture.
    Finally, B4) the secondary desiccation of the mixture obtained in B3) is carried out. It includes lowering the pressure to a value of 80 pbar or less, preferably the minimum possible value for the equipment, and raising the temperature of the shelves to a value between + 25 ° C and +35. ° C, preferably 25 ° C at a heating rate between 0.1 and 0.3 ° C / min, and keep it between 8 and 15 hours.
    In this way, a lyophilizate according to the invention is finally obtained.
    Preferably, the method according to the invention comprises, in step A), preliminary steps of treatment of the faecal microbiota sample, before mixing it with the diluent.
    Thus, preferably, step A) comprises the following substeps:
    Al) optionally, the preparation of a continuous gradient of iodixanol or 5- (N-2,3-dihydroxypropylacetamido) -2,4,6-triiodo-N, N'-bis (2,3 dihydroxypropyl) isophthalamide formed by freeze-thawing, A2) mixing at least one faecal microbiota sample of a donor subject with a saline buffer optionally comprising iodixanol or 5- (N-2,3-dihydroxypropylacetamido) -2 , 4, 6-triiododo-N, N'-bis (2,3-dihydroxypropyl) isophthalamide, anaerobically, A3) optionally, depositing the mixture obtained in A2) under the gradient obtained in
    Al), A4) sequential centrifugation with low acceleration in anaerobiosis, or ultracentrifugation of the mixture obtained in A2) or A3), A5) recovery of the bacterial ring or the supernatant formed at the end of step A4 ), in anaerobiosis, and A6) the mixture of the bacterial ring or the supernatant recovered in A5) with a diluent chosen from polyols, di- pentasaccharides, maltodextrins and mixtures thereof.
    In such a method, step A1), as well as de facto step A3), are optional.
    Preferably, the salt buffer used in the various substeps described above is an aqueous solution of HEPES comprising sodium chloride, preferably at a concentration of between 7 and 15 g / l. Preferably, HEPES is present in a concentration of between 8 and 50 mM, preferably between 9 and 42 mM.
    First alternative
    Preferably, according to a first alternative, the method according to the invention comprises the following steps:
    A1) the preparation of a continuous gradient of iodixanol or 5- (N-2,3-dihydroxypropylacetamido) -2,4,6-triiodo-N, N'-bis (2,3-dihydroxypropyl) isophthalamide formed by freeze-thawing, A2) mixing at least one faecal microbiota sample of a donor subject with a saline buffer supplemented with an aqueous solution comprising iodixanol or 5- (N-2,3-dihydroxypropylacetamido) -2,4,6-triiodo-N, N'-bis (2,3-dihydroxypropyl) isophthalamide, said salt buffer preferably being an aqueous solution of HEPES comprising sodium chloride, anaerobically, A3) deposition of the mixture obtained in A2) under the gradient obtained in A1), A4) the ultracentrifugation of the mixture obtained in A3), for a period of between 40 and 50 minutes, at a temperature of between 2 ° C. and 6 ° C., at an acceleration of between 13000 and 16000 × g, A5) the recovery of the bacterial ring formed at the end of step A4), in anaerobiosis, A 6) the mixture of the bacterial ring recovered in A5) with a diluent chosen from polyols, di-pentasaccharides, maltodextrins and mixtures thereof, and B) freezing the mixture obtained in A6) at a temperature below 50 ° C, preferably between -70 ° C and -100 ° C, and lyophilization. Stage A1) thus comprises the preparation of a continuous gradient of iodixanol or of 5- (N-2,3-dihydroxypropylacetamido) -2,4,6-triiododo-N, N'-bis (2, 3 dihydroxypropyl) isophthalamide formed by freeze-thawing.
    By continuous iodixanol gradient is meant a continuous gradient of iodixanol with a density ranging from 1.03 to 1.24, preferably ranging from 1.06 to 1.24.
    By continuous gradient of 5- (N-2,3-dihydroxypropylacetamido) -2,4,6-triiododo-N, N'-bis (2,3-dihydroxypropyl) isophthalamide is meant a continuous gradient of 5- (N) -2,3-dihydroxypropylacetamido) -2,4,6-triiodo-N, N'-bis (2,3-dihydroxypropyl) isophthalamide having a specific gravity ranging from 1.03 to 1.22.
    This step Al) is preferably carried out according to the following steps:
    Al.a) freezing a solution of iodixanol or 5- (N-2,3-dihydroxypropylacetamido) -2,4,6-triiodo-N, N'-bis (2,3-dihydroxypropyl) isophthalamide a temperature between -70 ° C and -100 ° C for at least 12 hours. Preferably, said solution is degassed. By solution of iodixanol or of degassed 5- (N-2,3-dihydroxypropylacetamido) -2,4,6-tri-iodo-N, N'-bis (2,3-dihydroxypropyl) isophthalamide is meant a solution of iodixanol or 5- (N-2,3-dihydroxypropylacetamido) -2,4,6-triiododo-N, N'-bis (2,3-dihydroxypropyl) isophthalamide in which the concentration of dissolved air is reduced, for example under vacuum. This makes it possible to create a micro-anaerobiosis favorable to the survival of the bacteria during the purification process; then
    Al.b) thawing of the solution obtained in Al.a) at room temperature for 2 to 4 hours, in order to obtain a continuous gradient of iodixanol or 5- (N-2,3-dihydroxypropylacetamido) -2,4 6-tri-iodo-N, N'-bis (2,3-dihydroxypropyl) isophthalamide.
    Preferably, the iodixanol solution used in step A1) is a solution having a concentration of between 15 and 25%, preferably around 20%, by weight of iodixanol by volume of solution (w / v). More preferably, the iodixanol solution used is obtained by diluting 3 times a commercial aqueous solution of iodixanol 60%, sold under the name OptiPrep (sterile aqueous solution 60% w / v of iodixanol), in a saline buffer preferably a buffer comprising 15mM HEPES and 9g / L NaCl having a pH of 7.0.
    Preferably, the solution of 5- (N-2,3-dihydroxypropylacetamido) -2,4,6-triiododo-N, N'-bis (2,3-dihydroxypropyl) isophthalamide is a solution comprising 20% by weight of 5- (N-2,3-dihydroxypropylacetamido) -2,4,6-triiododo-N, N'-bis (2,3-dihydroxypropyl) isophthalamide per volume of solution (w / v). More preferably, the 5- (N-2,3-dihydroxypropylacetamido) -2,4,6-triiododo-N, N'-bis (2,3-dihydroxypropyl) isophthalamide used is marketed by Progen Biotechnik under the name Nycodenz.
    Preferably, in step Al.a), the freezing of the solution of iodixanol or of 5- (N-2,3-dihydroxypropylacetamido) -2,4,6-tri-iodo-N, N'-bis (2,3 dihydroxypropyl) isophthalamide can be carried out for several days or even 1 month.
    Once the continuous gradient of iodixanol or 5- (N-2,3-dihydroxypropylacetamido) -2,4,6-triiodo-N, N'-bis (2,3 dihydroxypropyl) isophthalamide obtained in step Al), the subsequent steps A2), A4) and A5) are carried out anaerobically. Step A2) comprises mixing at least one fecal microbiota sample of a donor subject with a saline buffer supplemented with an aqueous solution comprising iodixanol or 5- (N-2,3-dihydroxypropylacetamido) -2,4,6-Triiodo-N, N'-bis (2,3-dihydroxypropyl) isophthalamide anaerobically. Preferably, said salt buffer is an aqueous solution of HEPES comprising sodium chloride. Preferably, HEPES is present in a concentration of between 30 and 50 mM in the saline buffer. Preferably, the sodium chloride is present at a concentration of between 7 and 15 g / l in the saline buffer. Preferably, the aqueous solution of iodixanol is at a concentration of between 50% and 70%, preferably about 60% (w / v). Preferably, the salt buffer: iodixanol aqueous solution ratio is about 1: 3.
    Preferably, the aqueous solution of 5- (N-2,3-dihydroxypropylacetamido) -2,4,6-triiododo-N, N'-bis (2,3-dihydroxypropyl) isophthalamide is at a concentration of 60% ( w / v). Preferably, the salt buffer: aqueous solution of 5- (N-2,3-dihydroxypropylacetamido) -2,4,6-triiodo-N, N'-bis (2,3 dihydroxypropyl) isophthalamide is approximately 1: 3.
    Thus, according to step A2), the salt buffer is premixed with an aqueous solution of iodixanol or 5- (N-2,3-dihydroxypropylacetamido) -2,4,6-tri-iodo-N, N ' bis (2,3 dihydroxypropyl) isophthalamide; thus, a saline buffer comprising iodixanol or 5- (N-2,3-dihydroxypropylacetamido) -2,4,6-triiodo-N, N'-bis (2,3-dihydroxypropyl) isophthalamide is obtained. Then the resulting mixture is mixed with the fecal microbiota sample.
    Preferably, the mixture of the fecal microbiota sample with a saline buffer comprising iodixanol or 5- (N-2,3-dihydroxypropylacetamido) -2,4,6-tri-iodo-N, N'- bis (2,3-dihydroxypropyl) isophthalamide is carried out in a ratio of 3 to 4 grams of sample for 22 to 30ml of saline buffer comprising iodixanol or 5- (N-2,3-dihydroxypropylacetamido) -2, 4,6-tri-iodo-N, N'-bis (2,3-dihydroxypropyl) isophthalamide.
    Preferably, when the fecal microbiota sample is filtered between steps A2) and A3), it undergoes a step of filtering the mixture obtained in A2) in anaerobiosis, in particular by using a Seward bag equipped with a filter.
    Then, the mixture obtained in A2), optionally filtered, is deposited under the gradient obtained in A1): this is step A3). Step A3) can be performed aerobically or anaerobically.
    Preferably, the mixture obtained in A2) is present in a syringe provided with a needle, and the gradient of iodixanol or 5- (N-2,3-dihydroxypropylacetamido) -2,4,6-tri-iodo N, N'-bis (2,3-dihydroxypropyl) isophthalamide is present in a tube-type container. In this case, the mixture obtained in A2) is deposited by immersing the needle of the syringe at the bottom of the tube containing the gradient of iodixanol or 5- (N-2,3-dihydroxypropylacetamido) -2,4,6 tri-iodo-N, N'-bis (2,3-dihydroxypropyl) isophthalamide, and emptying the contents of the syringe.
    Then, the preparation obtained in A3) is centrifuged under vacuum in a low-acceleration ultracentrifuge: this is step A4). This ultracentrifugation is carried out for a period of between 40 and 50 minutes, at a temperature of between 2 ° C. and 6 ° C., at a speed of between 13,000 and 16,000 × g. Preferably, the ultracentrifugation is carried out for a period of between 40 and 50 minutes, at a temperature of about 4 ° C, at a rate of about 14500-14600 x g. At the end of this step, a bacterial ring was formed within the gradient. This ring is recovered anaerobically in step A5). Π contains the microbiota of interest.
    Second alternative
    Preferably, according to a second alternative, the preparation method comprises the following steps: A2) the mixture of at least one sample of fecal microbiota of a donor subject with a saline buffer, under anaerobic conditions, A4) the sequential low-level centrifugation accelerating the mixture obtained in A2), in anaerobiosis, A5) recovering the supernatant formed at the end of step A4), in anaerobiosis, A6) the mixture of the supernatant recovered in A5) with a diluent chosen from the polyols, di-pentasaccharides, maltodextrins and mixtures thereof, and B) freezing the mixture obtained in A6) at a temperature below -50 ° C., preferably between -70 ° C. and -100 ° C., and then freeze-drying it .
    This second alternative comprises a low-speed sequential centrifugation step. By low acceleration, we mean an acceleration between 200 and 500 x g. Thus, the various particles present in the fecal microbiota sample (debris then bacterial cells) will sediment. Step A2) comprises mixing at least one faecal microbiota sample from a donor subject with an anaerobic phosphate buffer. Preferably, said salt buffer is an aqueous solution of HEPES comprising sodium chloride. Preferably, HEPES is present in a concentration of between 5 and 15 mM. Preferably, the sodium chloride is present at a concentration of between 7 and 15 g / l.
    Preferably, the mixture of the fecal microbiota sample with a saline buffer is carried out in a weight ratio of sample: buffer of 1:20 to 1:25.
    Preferably, when the fecal microbiota sample is filtered between steps A2) and A4), it undergoes a step of filtering the mixture obtained in A2) in anaerobiosis, in particular by using a Seward bag equipped with a filter.
    Then, the mixture obtained in A2), optionally filtered, is subjected to sequential centrifugation at low acceleration, anaerobically: this is step A4).
    The low-speed sequential centrifugation of step A4) is preferably carried out at an acceleration of between 200 and 500 x g for a time of between 5 and 15 minutes, at a temperature between 20 and 30 ° C. More preferably, the low-accelerated sequential centrifugation is carried out at an acceleration of about 300 x g for a time of between 5 and 15 minutes, at a temperature of between 20 and 25 ° C. At the end of this step, the supernatant is recovered anaerobically (step A5)). Π contains the microbiota of interest.
    Preferably, the low-accelerated sequential centrifugation of step d) and the step of recovering the subsequent supernatant e) are carried out several times, preferably at least twice, each step d) being carried out at an acceleration of between 200 and 500 xg for a time of between 5 and 15 minutes and at a temperature between 20 and 30 ° C.
    Thus, preferably, in step A5): A5.1) the supernatant formed at the end of step A4) is recovered, A5.2) the pellet obtained in A4) is mixed with a saline buffer (the same as that of step A2)), then subjected to low accelerated sequential centrifugation as described in step A4), and the supernatant thus obtained is mixed with the supernatant of fraction A5.1). This operation of washing the pellet can be repeated at will until exhaustion of the pellet in bacteria. In practice and for the sake of saving time and extraction volumes, a single washing of the pellet is typically practiced.
    Whatever the method according to the invention (first or second alternative), the latter may comprise the following stages, anaerobically, as step A6): a) centrifugation of the bacterial ring resuspended in a saline buffer or the supernatant obtained in A5), at an acceleration of between 3000 and 4000 × g for a time of between 5 and 15 minutes, at a temperature of between 20 and 30 ° C. b) recovery of the pellet obtained at the end of the step a), and resuspension in saline buffer, followed by centrifugation at an acceleration between 200 and 500 xg for a time of between 5 and 15 minutes, at a temperature between 20 and 30 ° C, c) recovery of supernatant obtained at the end of step b), and resuspended in a saline buffer, followed by centrifugation at an acceleration of between 3000 and 4000 × g for a time of between 5 and 15 minutes, at a temperature of between 20 and30 ° C, d) recovering the pellet obtained after step c), and e) mixing the pellet recovered in d) with a diluent chosen from polyols, di-pentasaccharides, maltodextrins and their mixtures.
    These steps a) to e) can thus be sub-steps of step A6). Steps a) to d) are intended to wash the microbiota obtained in A5). Preferably, the temperature of steps a) to d) is between 20 and 25 ° C.
    Preferably, in these steps a) to d), the saline buffer is an aqueous solution of HEPES comprising sodium chloride, preferably at a concentration of between 7 and 15 g / l. Preferably, HEPES is present in a concentration of between 8 and 15 mM.
    The pellet obtained in step d) contains the microbiota of interest. It can thus be mixed with the diluent as described in step e). At the end of step A5) or d) (regardless of the processes according to the first or second alternative), the recovered fraction is mixed with a diluent chosen from cryoprotectants such as polyols, di- pentasaccharides; bulking agents such as maltodextrins; and their mixtures.
    Then, step B) of freezing and lyophilization takes place, as described above.
    The present invention also relates to the use of a lyophilizate obtainable by the process according to the invention, as a research tool, especially as described above, in functional genomics, in metaproteomics or in immunology.
    The present invention also relates to a fecal microbiota lyophilizate of a donor subject that can be obtained by the method according to the invention, for its use in the transplantation of autologous or allogenic fecal microbiota. Indeed, the lyophilizate of fecal microbiota purified according to the method of the invention may be administered to the recipient patient.
    The recipient patient may be different from the donor subject, and the transplant is then allogenic.
    The recipient patient may also be identical to the donor subject, and the transplant is then autologous; this type of transplantation can take place when the subject, while healthy, gives a sample before the alteration of its microbiota. The lyophilizate is then preserved and then transplanted to the same subject (recipient patient) if the latter has in particular a Clostridium difficile infection. Autologous fecal microbiota transplantation has the advantage of avoiding transmission of pathogen from another donor.
    The present invention also relates to a fecal microbiota lyophilizate of a donor subject that can be obtained by the process according to the invention for its use for treating Clostridium difficile infections. The present invention also relates to a fecal microbiota lyophilizate of a donor subject that can be obtained by the method according to the invention, for its use to accompany the treatment or to treat a pathology selected from chronic inflammatory diseases of the subject. bowel (IBD), intestinal functional disorders, obesity, metabolic diseases and autoimmune diseases, allergies, neurological diseases and cancers. The present invention also relates to a lyophilizate obtainable by the process according to the invention, for its use to limit the side effects of a treatment chosen from antibiotic therapies, chemotherapies, radiotherapies and surgeries, in particular 'Digestive.
    The lyophilizate obtainable by the process according to the invention has a good viability of the bacteria present, as demonstrated in Example 4.
    Typically, the viability of fecal microbiota bacteria is measured using the LIVE / DEAD® fiacLight ™ Bacterial Viability Kit marketed by ThermoFisher Scientific. This kit makes it possible to distinguish living and dead bacteria on the basis of the integrity of their membranes, via two fluorophores, SYT09® and propidium iodide (PI). The first one penetrates into all the cells, honest or not, attaches to the DNA and emits at 540nm (green) after excitation at 470nm (blue laser). PI also targets DNA, but only enters cells with damaged membranes; it emits at 635nm (red) after excitation at 470nm. Such a kit may be associated with flow cytometry or epifluorescence microscopy.
    Preferably, the labeling of the bacteria with the mixture of the two SYT09® / PI fluorophores is carried out anaerobically. The invention will now be exemplified with the aid of the examples which follow, which are not limiting.
    EXAMPLE 1 Purification of a Fecal Microbiota Sample of a Donor Sure by a Continuous iodixanol Gradient (OptiPrep) According to the Invention
    Principle: Separation of the total bacterial fraction, by flotation within a continuous gradient of self-formed OptiPrep by freeze-thawing. Faecal dilution increased with OptiPrep is deposited under a continuous gradient of OptiPrep, preformed by freeze-thawing. During centrifugation, bacteria move up the gradient to their flotation density (1.110-1.190), while food and endogenous debris sink to the bottom of the gradient. The whole process is anerobiosis.
    Materials & Methods:
    Hepes-NaCl Buffers
    Preparation of the continuous gradients by freeze-thawing (step Al)): - pipette 16mL of degassed OptiPrep-20 solution, and transfer to a tube, avoiding aeration of the solution
    - freeze ovemight tubes at -80 ° C - thaw the tubes immobile and undisturbed at room temperature 2-3 hours before use; a continuous gradient of density 1.03-1.22 is automatically formed.
    Preparation of fecal dilutions in anaerobic chamber (step A2)): - Enter the stool sample into the anaerobic chamber - Weigh on the balance, on which is placed a sterile Seward double-fried sachet, the desired stool weight (maximum 3.5g for 1 gradient, possibly less, in this case make up to 3.5g with Hepes buffer 10mM-NaCl 9g / L) - Add in the Seward bag filter, 24mL of diluent for faecal suspensions - Homogenize the mixture
    transfer the filtered homogenized faecal suspension into a Falcon tube 50mL. From this tube, fill a 20mL syringe equipped with a needle with the faecal dilution. Achieving the gradient outside the anaerobic chamber (steps A3) and A4)); - Immerse the needle of the syringe obtained in A2) at the bottom of the preformed gradient by freezing-thawing obtained in Al) - Slowly load the 20mL faecal dilution under the preformed gradient - Repeat for other gradients
    - Weigh all the tubes and adjust exactly the same weight the tubes 2 to 2 with Hepes buffer 10mM-NaCl 9g / L - Carefully insert the tubes in the cold centrifuge pads for oscillating rotor - Centrifuge 45 min at 4 ° C 14,567 xg Recovery and washing the bacterial cells in an anaerobic chamber (steps A5). A6) and a) to d)): - Enter the centrifugation chambers into the anaerobic chamber without opening them - Open a stud - Using a pipette, remove the upper phase
    - Pipette the intermediate cell phase and distribute it in 2 tubes of 50mL Falcon - Add washing buffer (10mM Hepes-NaCl 9g / L) up to the 50th graduation of the 2 Falcon tubes - Take the same way the bacterial phases of all gradients - Centrifuge 10 min 4000 X g 22 ° C oscillating rotor - Eliminate the supernatant by suction - Add washing buffer at the ~ 25mF graduation of Falcon tubes
    - Resuspend the bacteria gently with a pipette; make up to 50mF - Centrifuge 5min 300 x g 22 ° C in oscillating rotor to remove residual debris - Transfer with a pipette the supernatant (containing the bacteria without residue) in 2 new Falcons 50mF; discard debris pellet - Centrifuge 10 min 3500 x g 22 ° C oscillating rotor - Remove supernatants
    Fe bacterial pellet without residue obtained can be resuspended in the chosen vehicle.
    Example 2 Purification of a Fecal Microbiota Sample from a Donor Subject by Low-Accelerated Sequential Centrifugation According to the Invention
    Principle: Separation of the total bacterial fraction, by low-speed sequential centrifugation.
    Materials & Methods:
    Hepes-NaCl buffer
    Preparation of fecal dilutions in anaerobic chamber: - Enter the stool sample into the anaerobic chamber - Transfer the desired weight of stool into the Seward bag filter - Complete QSP 350g with Hepes 10mM-NaCl 9g / L for 14g of stool ( a 1: 25 dilution) and homogenize (step b)
    - transfer 50mL of filtered homogenized faecal suspension into 6 tubes of Falcon 50mL - Centrifuge 10 min 300 xg 22 ° C in oscillating rotor to remove debris (step d)) - Divide the supernatants (they contain bacteria) in 12 new tubes 50mL (~ 25mL / tube) - Put the 6 pellets in suspension in 50mL Hepes 10mM-NaCl 9g / L and centrifuge for 10 min 300 xg 22 ° C in oscillating rotor (step d))
    - Divide the 6 new supernatants into the 12 pending tubes already containing the first supernatants; QSP 50mL for the 12 tubes with Hepes 10mM-NaCl 9g / L - Centrifuge the 12 tubes 10 min 3500 x g 22 ° C in oscillating rotor to pellet the bacteria - Using a pipette, gently remove the supernatant
    Finally, the bacterial pellets without residue obtained are resuspended in the vehicle chosen.
    EXAMPLE 3 Preparation of Fecal Microbiota Freeze-Dried Products Obtained in Examples 1 and 2
    The fractions obtained at the end of the processes of Examples 1 and 2 were mixed with the following diluents:
    - NaCl: 9g / L
    Maltodextrins: trehalose 15/5 or 5/15 in NaCl 9g / L1
    Iodixanol: 60% commercial aqueous solution diluted 3 times in HEPES 40mM-NaCl 9g / L buffer
    Then they were frozen at -80 ° C or -100 ° C
    Then they were subjected to the following lyophilization cycle:
    The secondary drying is carried out at +25.degree. C. at a pressure of 80 pbar for about 900 minutes.
    The lyophilizer was pre-cooled to -40 ° C prior to loading. Upon receipt of the samples, the vials stored in the dry ice were loaded into the apparatus and the lyophilizer was evacuated at the end of loading. Two PT 100 temperature probes were placed in two vials. The products being frozen before loading, the probes are above the product and not in the product.
    The duration of the cycle is 45 h. At the end of the cycle, the vials are capped under vacuum in the lyophilizer and capsulated after unloading.
    EXAMPLE 4 Viability of the Bacteria Present in the Fecal Microbiota Lyophilisates Obtained in Example 3
    Protocol:
    The viability of the bacteria present in the fecal microbiota lyophilisais obtained in Example 3 was measured according to the following protocol: successive decimal dilutions in anaerobic atmosphere, to finally be around 106 bacteria / mL in the sample intended for labeling ; this operation must be immediately following the resuspension of the bacterial pellets without residue in the chosen vehicle; the marking of the last dilution must also be immediate,
    Less than 30 min between resuspension and tagging, because live populations will grow rapidly if the vehicle contains a nutrient substrate: within 4 hours of waiting at room temperature, anaerobically and in the presence of a single nutrient substrate, the population increases by half a log and the percentage of live bacteria increases by 10%, labeling of bacteria, using the LIVE / DEAD® SacLight ™ Bacterial Viability Kit according to the manufacturer's instructions, - quantification of live and dead bacteria in cytometry. flux: the time between the marking and the quantification does not exceed 20 minutes, that the marked samples wait (always protected from the light) at ambient temperature or on crushed ice. Results:
    The results are shown in the table below:
    Thus, the results show that the viability of the bacteria present in the purified samples according to the invention is very good when the bacteria are lyophilized with maltodextrins.
    EXAMPLE 5 Obtaining and Viability of Bacteria Present in Unpurified Fecal Microbiota Lyophilisates
    A microbiota was collected and suspended in the following solutions:
    - NaCl: 9g / L
    Maltodextrins: trehalose 15/5 or 5/15 in NaCl 9g / L1
    The suspensions were then frozen at -80 ° C. or -100 ° C. They were then subjected to the following lyophilization cycle:
    The lyophilizer was pre-cooled to -45 ° C prior to loading. Upon receipt of the samples, the products stored in the dry ice were loaded into the apparatus and the lyophilizer was evacuated at the end of loading.
    The cycle time is 48 h. At the end of the cycle, the vials are capped under vacuum in the lyophilizer and capsulated after unloading.
    The quality of the bacterial populations was evaluated in terms of diversity via DNA extraction and its analysis by sequencing the rDNA 16S gene. A phylogenetic analysis was then conducted to establish the profiles of the different samples in order to compare them. The results are shown in the table below:
    Thus, the results show that the correlation levels between the taxonomic profiles are very high for the 3 formulations, demonstrating that the process effectively retains about 90% of the bacterial populations.
    The viability was evaluated in the same way as shown in Example 4 and the results after 10 months of storage are presented below:
    After 10 months, the suspensions carried out with NaCl have viabilities significantly lower than the other 2.
    Thus, the microbiota obtained according to the method make it possible both to preserve the bacterial populations since the correlations of the phylogenetic profiles are very high, but they also make it possible to keep the bacteria alive longer than the NaCl formulation.
    权利要求:
    Claims (11)
    [1" id="c-fr-0001]
    A process for preparing a fecal microbiota lyophilizate of a donor subject, comprising the steps of: A) mixing a fecal microbiota sample of a donor subject with a diluent selected from polyols, diols and the like. pentasaccharides, maltodextrins and mixtures thereof, and B) freezing the mixture obtained in A) at a temperature below -50 ° C, preferably between -70 ° C and -100 ° C, and lyophilization.
    [2" id="c-fr-0002]
    2. Method according to claim 1, characterized in that step A) comprises the following steps: A1) optionally, the preparation of a continuous gradient of iodixanol or 5- (N-2,3-dihydroxypropylacetamido) 2, 4, 6-triiodo-N, N'-bis (2,3-dihydroxypropyl) isophthalamide formed by freezing-thawing, A2) mixing at least one faecal microbiota sample of a donor subject with a buffer saline optionally comprising iodixanol or 5- (N-2,3-dihydroxypropylacetamido) -2,4,6-triiododo-N, N'-bis (2,3-dihydroxypropyl) isophthalamide, anaerobically, A3) optionally, the deposition of the mixture obtained in A2) under the gradient obtained in A1), A4) the sequential centrifugation with low acceleration in anaerobiosis, or the ultracentrifugation of the mixture obtained in A2) or A3), A5) the recovery of the bacterial ring or the supernatant formed at the end of step A4), in anaerobiosis, A6) the mixture of the bacterial ring or the supernatant recovered at A5) with a diluent chosen from polyols, di- pentasaccharides, maltodextrins and mixtures thereof.
    [3" id="c-fr-0003]
    3. Method according to claim 1 or 2, characterized in that it comprises, as step A6), the following steps in anaerobiosis: a) centrifugation of the bacterial ring resuspended in a saline buffer or the supernatant obtained in A5 ), at an acceleration of between 3000 and 4000xg for a time of between 5 and 15 minutes, at a temperature of between 20 and 30 ° C, b) recovery of the pellet obtained at the end of step a), and delivery in suspension in a saline buffer, then centrifugation at an acceleration between 200 and 500xg for a time of between 5 and 15 minutes, at a temperature between 20 and 30 ° C, c) recovery of the supernatant obtained at the end of the step b), and resuspension in a saline buffer, followed by centrifugation at an acceleration between 3000 and 4000xg for a time of between 5 and 15 minutes, at a temperature between 20 and 30 ° C, d) recovery of the pellet got after step c), and e) mixing the pellet recovered in d) with a diluent selected from polyols, di- to pentasaccharides, maltodextrins and mixtures thereof.
    [4" id="c-fr-0004]
    4. Method according to one of claims 1 to 3, characterized in that the lyophilization of step B) is performed under the following conditions: Bl) freezing the mixture obtained in A) at a temperature below -50 ° C , preferably between -70 ° C and -100 ° C, B2) the loading of the frozen mixture obtained in B1) in a lyophilizer pre-cooled at a temperature between -50 ° C and -30 ° C, at atmospheric pressure, then B3) at least one primary drying step of the feed mixture B2) comprising lowering the pressure to a value between 80 and 200 pbar, then increasing the temperature of the shelves to a value between -20 ° C and + 25 ° C, applying a heating rate of between 0.2 and 0.5 ° C / min, then B4) the secondary drying of the mixture obtained in B3) comprising lowering the pressure at a value less than or equal to 80 pbar, and the rise in temperature shelves at a value between + 25 ° C and + 35 ° C, at a heating rate between 0.1 and 0.3 ° C / min, and keep it there for 8 to 15 hours.
    [5" id="c-fr-0005]
    5. Method according to one of claims 2 to 4, characterized in that the salt buffer is an aqueous solution of HEPES comprising sodium chloride, preferably at a concentration between 7 and 15g / l.
    [6" id="c-fr-0006]
    6. Method according to one of claims 1 to 5, characterized in that it comprises the following steps: A1) the preparation of a continuous gradient iodixanol or 5- (N-2,3-dihydroxypropylacetamido) - 2, 4, 6-triiodo-N, N'-bis (2,3-dihydroxypropyl) isophthalamide formed by freezing-thawing, A2) mixing at least one faecal microbiota sample of a donor subject with a buffer saline supplemented with an aqueous solution comprising iodixanol or 5- (N-2,3-dihydroxypropylacetamido) -2,4,6-triiodo-N, N'-bis (2,3-dihydroxypropyl) isophthalamide, said salt buffer being preferably an aqueous solution of HEPES comprising sodium chloride, anaerobically, A3) depositing the mixture obtained in A2) under the gradient obtained in A1), A4) ultracentrifugation of the mixture obtained in A3) for a period of between 40 and 50 minutes at a temperature of between 2 ° C and 6 ° C at a speed between 13000 and 16000 x g, A5) recovering the bacterial ring formed at the end of step A4), in anaerobiosis, A6) the mixture of the bacterial ring recovered in A5) with a diluent chosen from polyols, diols and pentasaccharides, maltodextrins and mixtures thereof, and B) freezing the mixture obtained in A6) at a temperature below -50 ° C, preferably between -70 ° C and -100 ° C, and lyophilization.
    [7" id="c-fr-0007]
    7. Method according to one of claims 1 to 6, characterized in that the preparation of a continuous gradient of iodixanol formed by freeze-thawing step Al) is carried out according to the following steps: Al.a) freezing of a solution of iodixanol or 5- (N-2,3-dihydroxypropylacetamido) -2,4,6-triiododo-N, N'-bis (2,3-dihydroxypropyl) isophthalamide at a temperature between - 70 ° C and -100 ° C for at least 12 hours; then Al.b) thawing of the solution obtained in Al.a) at room temperature for 2 to 4 hours, in order to obtain a continuous gradient of iodixanol or 5- (N-2,3-dihydroxypropylacetamido) -2, 4,6-tri-iodo-N, N'-bis (2,3-dihydroxypropyl) isophthalamide.
    [8" id="c-fr-0008]
    8. Method according to one of claims 1 to 5 or 7, characterized in that it comprises the following stages in anaerobiosis: A2) the mixture of at least one sample of fecal microbiota of a donor subject with a saline buffer , A4) the low-accelerated sequential centrifugation of the mixture obtained in A2), A5) the recovery of the supernatant formed at the end of step A4), A6) the mixture of the supernatant recovered in A5) with a diluent chosen from polyols, di- pentasaccharides, maltodextrins and mixtures thereof, and B) freezing the mixture obtained in A6) at a temperature below -50 ° C, preferably between -70 ° C and -100 ° C, and its lyophilization.
    [9" id="c-fr-0009]
    9. Process according to claim 8, characterized in that the low accelerated sequential centrifugation of step A4) is carried out at an acceleration of between 200 and 500 × g for a time of between 5 and 15 minutes, at a temperature between and 30 ° C.
    [10" id="c-fr-0010]
    10. Fecal microbiota lyophilisate of a donor subject obtainable by the method according to one of claims 1 to 9, for its use in the transplantation of autologous or allogenic fecal microbiota, or for treating intestinal dysbiosis.
    [11" id="c-fr-0011]
    11. Use of a lyophilizate obtainable by the method according to one of claims 1 to 9, as a research tool, preferably in functional genomics, metaproteomics or immunology.
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    同族专利:
    公开号 | 公开日
    CN108430481B|2021-12-28|
    PL3389679T3|2020-09-07|
    CA3007289A1|2017-06-22|
    AU2016370600A1|2018-06-28|
    WO2017103550A1|2017-06-22|
    ES2777624T3|2020-08-05|
    KR102341203B1|2021-12-20|
    US10736849B2|2020-08-11|
    HUE048991T2|2020-09-28|
    IL259888A|2020-08-31|
    AU2016370600B2|2021-07-15|
    PT3389679T|2020-04-21|
    FR3045383B1|2019-06-14|
    IL259888D0|2018-07-31|
    KR20180086506A|2018-07-31|
    CN108430481A|2018-08-21|
    US20180369147A1|2018-12-27|
    EP3389679B1|2020-02-12|
    EP3389679A1|2018-10-24|
    JP2019501156A|2019-01-17|
    DK3389679T3|2020-03-16|
    JP6948321B2|2021-10-13|
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    优先权:
    申请号 | 申请日 | 专利标题
    FR1562750A|FR3045383B1|2015-12-18|2015-12-18|PROCESS FOR THE FREEZING OF A FECAL MICROBIOTE SAMPLE|
    FR1562750|2015-12-18|FR1562750A| FR3045383B1|2015-12-18|2015-12-18|PROCESS FOR THE FREEZING OF A FECAL MICROBIOTE SAMPLE|
    PL16826115T| PL3389679T3|2015-12-18|2016-12-19|Method for lyophilisation of a sample of fecal microbiota|
    EP16826115.4A| EP3389679B1|2015-12-18|2016-12-19|Method for lyophilisation of a sample of fecal microbiota|
    AU2016370600A| AU2016370600B2|2015-12-18|2016-12-19|Method of lyophilization of a sample of faecal microbiota|
    JP2018531198A| JP6948321B2|2015-12-18|2016-12-19|Freeze-drying method for stool bacterial flora sample|
    DK16826115.4T| DK3389679T3|2015-12-18|2016-12-19|Method for lyophilizing a sample of fecal microbiota|
    PT168261154T| PT3389679T|2015-12-18|2016-12-19|Method for lyophilisation of a sample of fecal microbiota|
    US16/063,419| US10736849B2|2015-12-18|2016-12-19|Method of lyophilization of a sample of faecal microbiota|
    PCT/FR2016/053550| WO2017103550A1|2015-12-18|2016-12-19|Method for lyophilisation of a sample of fecal microbiota|
    KR1020187020068A| KR102341203B1|2015-12-18|2016-12-19|Method of freeze-drying samples of fecal microbiota|
    CA3007289A| CA3007289A1|2015-12-18|2016-12-19|Method for lyophilisation of a sample of fecal microbiota|
    HUE16826115A| HUE048991T2|2015-12-18|2016-12-19|Method for lyophilisation of a sample of fecal microbiota|
    CN201680073680.7A| CN108430481B|2015-12-18|2016-12-19|Method for freeze-drying fecal microbiota samples|
    ES16826115T| ES2777624T3|2015-12-18|2016-12-19|Freeze-drying process of a fecal microbiota sample|
    IL259888A| IL259888A|2015-12-18|2018-06-07|Method of lyophilization of a sample of faecal microbiota|
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