Bacterial compositions and methods for growing bacteria on particles - Patents.com

Abstract

The present invention is directed inter alia to a composition comprising a population of at least one bacterial strain at least partially attached to a particle, the particle comprising (i) a polysaccharide-based material and (ii) a metal carbonate. Further provided herein are uses of said composition, e.g. for preventing or treating dysbiosis, and methods for its preparation.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 327,455, entitled "Compositions and Methods for Growing Bacteria on Particles," filed April 5, 2022, the contents of which are incorporated by reference in their entirety herein.

[0002] The present invention is in the field of microbiology, and in particular relates to bacterial compositions in attached and / or aggregated form, and methods of preparing and using same. [Background technology]

[0003] The human body harbors a rich and diverse microbial community, creating a network of bacteria-human cell interactions that profoundly influences our health and even our behavioral patterns.

[0004] There is growing evidence supporting the use of probiotics to promote human health, for example by benefiting the immune system and suppressing infections.

[0005] A wide variety of bacterial compositions have been developed to prevent or treat a variety of diseases, including chronic disorders such as dysbiosis, obesity, infections, colitis, inflammatory bowel disease (such as Crohn's disease), autoimmune diseases, cancer, poor vaginal health, and various skin conditions.

[0006] However, due to the harsh environmental conditions during migration to and at the site of interest, one of the challenges in the delivery of biological therapeutics is the successful survival, colonization and expansion of the delivered bacteria.

[0007] Several attempts have been made to develop bacterial compositions better suited to achieve bacterial colonization and sustain beneficial effects, each of the known compositions and methods having certain advantages and disadvantages. Summary of the Invention [Problem to be solved by the invention]

[0008] There remains an unmet need for alternative compositions that can overcome the shortcomings of existing compositions and provide advantageous methods for preventing or treating diseases and / or conditions, such as compositions comprising bacteria attached to alternative particles.

[0009] Advantageously, such replacement particles can be at least partially dissolved during preparation of the composition while maintaining the viability and beneficial effects of the bacteria, thereby enabling the production of compositions with reduced contents of inactive materials and increased relative contents of bacteria in the final product. [Means for solving the problem]

[0010] In some aspects of the present invention, a composition is provided comprising a population of at least one bacterial strain at least partially attached to a particle, the particle comprising (i) a polysaccharide-based material and (ii) a metal carbonate.

[0011] In another aspect of the invention, there is provided a method for preparing a composition comprising a population of at least one bacterial strain at least partially attached to particles, the particles comprising (i) a polysaccharide-based material and (ii) a metal carbonate, the method comprising the steps of providing the population of at least one bacterial strain, providing particles comprising (i) a polysaccharide-based material and (ii) a metal carbonate, contacting the population of at least one bacterial strain with the particles, wherein the contacting is performed in a solution, and allowing the population of at least one bacterial strain to at least partially adhere to the particles, thereby preparing a composition comprising the population of at least one bacterial strain at least partially attached to the particles.

[0012] In some embodiments, the particles are water-insoluble particles.

[0013] In some embodiments, the metal carbonate is not produced by the at least one bacterial strain.

[0014] In some embodiments, the composition has at least one characteristic selected from: (a) comprising particles in the form of a composite comprising the polysaccharide-based material and the metal carbonate; and (b) comprising each of the polysaccharide-based material and the metal carbonate within different or separate particles.

[0015] In some embodiments, the particles have at least one characteristic selected from: (a) being in the form of a composite comprising the polysaccharide-based material and the metal carbonate; and (b) comprising each of the polysaccharide-based material and the metal carbonate within separate particles.

[0016] In some embodiments, at least a portion of the particles are in the form of a composite comprising (i) a polysaccharide-based material and (ii) a metal carbonate, hi some embodiments, a portion of the particles comprise a polysaccharide-based material and a metal carbonate that are not combined together within the same particle.

[0017] In some embodiments, each particle may include one or more additional materials, hi some embodiments, the particles include at least one additional material.

[0018] In some embodiments, the additional material is or includes dicalcium phosphate (DCP).

[0019] In some embodiments, the particles further comprise dicalcium phosphate (DCP).

[0020] In some embodiments, the compositions and / or methods further comprise particles that are devoid of polysaccharide-based material and metal carbonate.

[0021] In some embodiments, the at least one bacterial strain is attached to at least one of the surface of the polysaccharide-based material and the surface of the metal carbonate.

[0022] In some embodiments, the particles include more than one type of polysaccharide-based material.In some embodiments, the particles include more than one type of metal carbonate.

[0023] In some embodiments, the polysaccharide-based material is or includes a cellulose derivative.

[0024] In some embodiments, the polysaccharide-based material is or comprises microcrystalline cellulose (MCC).

[0025] In some embodiments, the metal carbonate is or includes calcium carbonate.

[0026] In some embodiments, the composition comprises a metal carbonate at a concentration of no greater than 85% (w / w) of the total weight of the particles in the composition, based on the dry weight of the particles.

[0027] In some embodiments, the composition is characterized by one of: (i) comprising a metal carbonate in a concentration of at least 15% (w / w) of the total weight of particles in the composition, based on the dry weight of the particles; or (ii) further comprising metal phosphate-containing particles, wherein the metal phosphate and metal carbonate constitute at least 15% (w / w) of the total weight of particles in the composition, based on the dry weight of the particles.

[0028] In some embodiments, the w / w ratio of the polysaccharide-based material to the metal carbonate is 1:10 to 10:1 based on the dry weight of the particle. In some embodiments, the w / w ratio of the polysaccharide-based material to the metal carbonate is 1:6 to 6:1 based on the dry weight of the particle.

[0029] In some embodiments, the composition comprises a trace amount of a metal carbonate.

[0030] In some embodiments, the composition further comprises bacteria that are not attached to particles, hi some embodiments, the composition further comprises planktonic bacteria.

[0031] In some embodiments, the composition comprises two or more bacterial strains.

[0032] In some embodiments, the composition comprises a trace amount of a metal carbonate.

[0033] In some embodiments, the composition is provided in a solid form.

[0034] In some embodiments, the composition further comprises a pharma- ceutically acceptable carrier or excipient.

[0035] In some embodiments, the composition is obtained by a process comprising contacting, mixing, incubating and / or culturing a population of at least one bacterial strain with particles, and at least partially attaching the bacteria to the particles.

[0036] In some embodiments, the composition is obtained by a process comprising culturing a population of at least one bacterial strain in the presence of the particles.

[0037] In some embodiments, the present invention provides methods for preparing the compositions disclosed herein.

[0038] In some embodiments, at least partially attaching the population of at least one bacterial strain to the particles comprises culturing the population with the particles.

[0039] In some embodiments, at least partially attaching the population of at least one bacterial strain to the particles comprises culturing the population with the particles for a period of time sufficient for the population to attach to the particles, for example, between 1 hour and 15 days.

[0040] In some embodiments, at least partially attaching the population of at least one bacterial strain to the particles comprises culturing the population with the particles until the population attaches to the particles, for example for 1 hour to 15 days.

[0041] In some embodiments, the method further comprises the step of mixing the population of at least one bacterial strain with the particles in a dry form prior to the contacting step.

[0042] In some embodiments, the contacting comprises mixing, incubating and / or culturing. In some embodiments, inoculation is the first step of the culturing step and is performed in the presence of the particles. In some embodiments, the contacting is performed in a solution selected from the group consisting of saline, phosphate buffered saline and growth medium. In some embodiments, the solution is a growth medium. In some embodiments, different steps of the method can be performed in the same solution or in different solutions. In some embodiments, the contacting comprises culturing a population of at least one bacterial strain in a growth medium containing the particles.

[0043] In some embodiments, the methods include contacting two or more bacterial strains with the particles.

[0044] In some embodiments, at least a portion of the particles used in the methods of the invention are in the form of a composite comprising a polysaccharide-based material and a metal carbonate, hi some embodiments, at least a portion of the particles used in the methods of the invention comprise a polysaccharide-based material and a metal carbonate that are not combined together within the same particle.

[0045] In some embodiments, during the contacting step, the particles have at least one characteristic selected from: (a) being in the form of a composite comprising the polysaccharide-based material and the metal carbonate; and (b) comprising each of the polysaccharide-based material and the metal carbonate within different or separate particles.

[0046] In some embodiments, the particles provided include at least one additional material.

[0047] In some embodiments, the weight of metal carbonate relative to the volume of solution ranges from 9 g per liter to 215 g per liter, based on the dry weight of the particles.

[0048] In some embodiments, the total weight of particles provided relative to the volume of solution ranges from 60 g per liter to 250 g per liter, based on the dry weight of the particles.

[0049] In some embodiments, the particles provided include metal carbonate at a concentration of 85% (w / w) or less of the total weight of the particle, based on the dry weight of the particle.

[0050] In some embodiments, the particles provided are characterized by either one of: (i) comprising a metal carbonate in a concentration of at least 15% (w / w) of the total weight of the particle, based on the dry weight of the particle; or (ii) further comprising a metal phosphate, wherein the metal phosphate and metal carbonate constitute at least 15% of the total weight of the particle, based on the dry weight of the particle.

[0051] In some embodiments, the method further comprises the step of: 2 or both, thereby removing metal carbonate from the particles.

[0052] In another aspect of the invention, a method for treating a patient is disclosed, the method comprising administering to the patient a composition of the invention.

[0053] According to another aspect, there is provided a method for preventing or treating a dysbiosis in a subject in need thereof, comprising administering to the subject an effective amount of a composition of the invention.

[0054] In some embodiments, the present invention provides a method for preparing a composition disclosed herein, comprising inoculating a solution with a population comprising at least one bacterial strain, and culturing the population comprising at least one bacterial strain in the solution comprising particles for a time sufficient for the bacteria to at least partially adhere to the particles, wherein the particles comprise (i) a cellulosic material and (ii) a metal carbonate.

[0055] According to another aspect, there is provided a composition produced according to the inventive methods disclosed herein.

[0056] According to another aspect, there is provided a pharmaceutical composition comprising the composition of the invention disclosed herein and an acceptable carrier.

[0057] In some embodiments, the pharmaceutical composition is for use in treating or preventing a dysbiosis in a subject in need thereof.

[0058] In some embodiments, the composition further comprises an acceptable carrier. In some embodiments, the composition is for use in treating or preventing dysbiosis in a subject in need thereof. In some embodiments, the composition is for agricultural or veterinary use.

[0059] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice or test embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, this patent specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not necessarily intended to be limiting.

[0060] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. It should be understood, however, that these detailed descriptions and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various modifications and changes within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0061] [Figure 1] 1 is a bar graph showing the bacterial count (Log CFU / ml) of Bifidobacterium bifidum after cultivation in planktonic growth form (in the absence of particles), the bacterial count in the combined particle-attached and non-attached bacterial fractions after cultivation in the presence of particles (labeled "Group 1"), the bacterial count in the particle-attached bacterial fraction alone (labeled "Group 2"), and the bacterial count after partial removal of metal carbonate particles from the particle-attached fraction with acid, i.e., after removal of the metal carbonate particles from the Group 2 composition (labeled "Group 3"). [Diagram 2] Figure 1 is a bar graph showing the survival rate of wet (end of culture) Lactobacillus plantarum cultures after exposure to a series of increasing H2O2 concentrations. The resistance of attached bacterial fractions prepared by different culture methods was tested, namely (i) cultured in the presence of a CaCO3:MCC particle mixture (1:1 w / w ratio) according to an embodiment of the invention, and (ii) bacteria cultured at 96 w / p. Planktonic growth morphology was used as reference. Total bacterial counts (CFU) were measured after H2O2 exposure. Results are expressed as a percentage compared to control untreated bacteria of each group ("no H2O2 added" was considered as 100%). [Diagram 3]1 is a bar graph showing the survival of freeze-dried Lactobacillus plantarum cultures after reconstitution and exposure to a series of increasing H2O2 concentrations. Resistance was tested for adherent bacterial fractions and planktonic growth forms prepared by culturing in the presence of CaCO3:MCC particle mixtures (1:1 w / w ratio) according to an embodiment of the invention. Total bacterial counts (CFU) were measured after H2O2 stress. Results are expressed as a percentage compared to control untreated bacteria for each group ("no H2O2 added" was considered as 100%). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] According to some embodiments, a composition is provided that comprises a population comprising at least one bacterial strain at least partially attached to a particle.

[0063] According to some embodiments, there is provided a composition comprising a population comprising at least one bacterial strain grown in the presence of particles as detailed herein.

[0064] According to some embodiments, a method is provided for producing a composition comprising a population comprising at least one bacterial strain at least partially attached to a particle.

[0065] According to some embodiments, methods are provided for increasing bacterial adhesion to particles, e.g., when using particles comprising (i) a polysaccharide-based material and (ii) a metal carbonate, as compared to when the particles comprising the polysaccharide-based material are used in the absence of the metal carbonate particles.

[0066] According to some embodiments, there is provided a method for growing a population comprising at least one bacterial strain (e.g. gut-derived bacteria, skin-derived bacteria, vaginal-derived bacteria), comprising mixing, contacting and / or culturing in the presence of particles as detailed herein.

[0067] In some embodiments, the particles are added, mixed, contacted (e.g., incubated) and / or cultured with a population of at least one bacterial strain in a solution such as saline, phosphate buffer, growth medium, etc. In some embodiments, the population of at least one bacterial strain and the particles are contacted when the bacteria and / or particles are in a wet or hydrated form. In some embodiments, the population and the particles are first contacted in a dry or non-hydrated form, e.g., after being subjected to a drying process, and then a solution is added and contacting or culturing is performed. In some embodiments, either the population or the particles are in a wet form and the other is in a dry form. In some embodiments, the particles are provided in a dry form.

[0068] In some embodiments, the solutions include aqueous and liquid solutions.

[0069] The methods and compositions of the invention, in some embodiments, have several advantageous properties (e.g., increased bacterial adhesion to particles, increased bacterial load, and / or increased ability to adhere to mucosal surfaces), as demonstrated and detailed below. In some embodiments, particle-attached bacteria prepared according to the invention are resistant to environmental stresses and persistent under unfavorable conditions in a variety of environments. In some embodiments, particle-attached bacteria prepared according to the invention are capable of and / or configured to adhere to surfaces, such as to enable successful bacterial colonization in a subject to which they are administered.

[0070] In some embodiments, the compositions, when used or cultured in the presence of particles, e.g., as detailed herein, are characterized as having an increased number of bacteria attached to the particles compared to culture in the presence of control particles (e.g., particles comprising microcrystalline cellulose) (in the absence of metal carbonate particles). In some embodiments, the increase in bacterial attachment is in the range of 1.1-50 fold, e.g., at least 1.1 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 45 fold bacterial attachment, or any value and range therebetween, compared to the control. Each possibility represents a separate embodiment of the present invention.

[0071] In some embodiments, the terms "bacteria," "polysaccharide-based materials," "metal carbonates," and "metal phosphates," as used throughout this specification, include singular and plural referents unless the context clearly indicates otherwise.

[0072] In some embodiments, the term "a population of at least one bacterial strain" encompasses a population of at least one bacterial strain and a population of two or more bacterial strains.

[0073] In some embodiments, the terms "attached" and "adhered" with respect to interactions between bacteria and particles are interchangeable and refer to the adsorption of bacteria to surfaces / particles, e.g., by weak and / or strong interactions, e.g., by flagella, pili, lipopolysaccharides, exopolysaccharides, collagen-binding adhesive proteins, etc.

[0074] In some embodiments, the population of at least one bacterial strain adheres to the particles during cultivation. In some embodiments, the population of at least one bacterial strain is grown in the presence of the particles.

[0075] In some embodiments, the invention provides a method for increasing the amount of bacteria when used or cultured in the presence of particles, e.g., as detailed herein, compared to use or culture in the presence of particles comprising microcrystalline cellulose and in the absence of metal carbonate particles, and / or compared to planktonic culturing in the absence of particles. In some embodiments, the increase in bacterial amount at the end of the culture is in the range of 1.1 to 50 times, e.g., at least 1.1 times, at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 5.5 times, at least 6 times, at least 6.5 times, at least 7 times, at least 7.5 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 45 times the amount of bacteria, or any value and range therebetween, compared to use or culture in the presence of particles comprising microcrystalline cellulose and in the absence of metal carbonate particles, and / or compared to planktonic culturing. Each possibility represents a separate embodiment of the present invention.

[0076] In some embodiments, compositions according to the invention are advantageously mucoadhesive, e.g., CaCO, which has the ability to be bound and / or retained on mucosal surfaces, such as by interaction with mucin. 3 In some embodiments, the compositions of the present invention can and / or are configured to adhere to mucosal surfaces (e.g., epithelial cells of the skin, intestine, or vagina), for example, to enable successful bacterial colonization in a subject to which they are administered. In some embodiments, the compositions are configured to bind and / or be retained on mucosal surfaces and release bacteria from the composition over an extended period of time at the target area. For example, bacteria beneficial for treating the skin, vagina, or intestine can be included in the composition, and the bacteria can be retained and released at the target area without being washed away, for maximum therapeutic benefit.

[0077] In some embodiments, the method of the invention includes a step of partial or complete removal / decomposition / dissolution of metal carbonate particles. In some embodiments, the viability and beneficial effects, e.g., functionality, of the bacteria are substantially maintained after this removal or decomposition step. In some such embodiments, the composition has a reduced content of inert materials and an increased content of bacteria, e.g., compared to the ratio before the removal step. Typically, the term "inert materials" refers to materials that have no biological activity for the intended use of the composition. In some embodiments, substantially maintaining the viability, beneficial effects, and / or functionality of the bacteria refers to maintaining at least 80% of the initial value before the removal / decomposition step. In some embodiments, 80%-100% of the initial value before the removal / decomposition step is maintained.

[0078] In some embodiments, bacterial viability is determined by any method known to one of skill in the art, such as, but not limited to, viable colony forming units (CFU), live-dead staining, propidium monoazide qPCR (PMA-qPCR), metabolic assays, spectrophotometry, or any combination thereof. Methods for determining bacterial viability are common, such as those disclosed herein, and will be apparent to one of skill in the art.

[0079] In some embodiments, the beneficial effect is determined by measuring the ability of the bacteria to affect the metabolic processes of a subject and / or ameliorate any disease, disorder or condition in a subject, measuring the potential metabolic pathways / pathways of the bacteria, e.g., their potential to produce, synthesize, consume and / or utilize certain metabolites / organic compounds.

[0080] In some embodiments, the present invention also provides methods for increasing the resistance of bacteria, e.g., compared to bacterial growth in other growth forms, as well as compositions comprising resistant bacteria, e.g., antibiotic-resistant, acid-resistant, heat-resistant, and cold-resistant bacteria, that can withstand harsh environments and extreme manufacturing conditions.

[0081] In some embodiments, the compositions disclosed herein are administered to a subject in need thereof prior to, concomitantly with, together with, or after administration of an antibiotic and / or an acidic substance. In some embodiments, the resilient / resistant bacterial population prepared according to the present invention is co-administered or co-treated with an antibiotic and / or an acidic substance. In some embodiments, the antibiotic / acidic substance is released or administered prior to the beneficial / probiotic bacteria, thereby reducing the abundance of pathogens in the target tissue and contributing to the successful colonization of the delivered beneficial or probiotic bacteria. Such combined treatment may be advantageous, for example, in preventing or treating conditions of the female urogenital tract characterized by elevated pH levels and / or increased abundance of pathogenic bacteria. One non-limiting example of such a condition is bacterial vaginosis, in which administration of lactic acid-producing bacteria prepared according to the present invention with or without an antibiotic and / or an acidic substance may be beneficial. Thus, in some embodiments, the compositions of the present invention are used in the treatment of bacterial vaginosis with or without an antibiotic and / or an acidic substance. Antibiotics and / or acidic substances can be co-administered with the compositions of the present invention simultaneously, sequentially, or alternately. In some embodiments, the antibiotic is any antibiotic used in the treatment of bacterial vaginosis. Non-limiting examples of antibiotics include metronidazole (Flagyl), clindamycin (Cleocin) and metronidazole. In some embodiments, the acidic substance comprises a pH adjusting agent. Non-limiting examples of pH adjusting agents of the present invention are sodium bicarbonate, ascorbic acid, citric acid, acetic acid, fumaric acid, propionic acid, malic acid, succinic acid, gluconic acid, tartaric acid, lactic acid, boric acid cranberry extract, and any combination thereof.

[0082] In some embodiments, extreme manufacturing conditions are, for example, temperatures greater than 37°C, e.g., greater than 38°C, greater than 39°C, greater than 40°C, greater than 45°C, greater than 50°C, temperatures in the range of greater than 37°C to 40°C, greater than 37°C to 45°C, greater than 37°C to 50°C, temperatures in the range of 38°C to 50°C, or temperatures in the range of 50°C to 55°C.

[0083] In some embodiments, the composition comprises a population of at least one bacterial strain at least partially attached to a particle, where the particle comprises (i) a polysaccharide-based material and (ii) a metal carbonate.

[0084] In some embodiments, the composition and / or method includes multiple particle types or particle forms. In some embodiments, the composition and / or method includes, e.g., upon contact or incubation, at least one of: (a) particles in the form of a composite including a polysaccharide-based material and a metal carbonate, and (b) particles including only one of (i) a polysaccharide-based material or (ii) a metal carbonate. By "particles including only one of (i) a polysaccharide-based material or (ii) a metal carbonate" is meant that the composition or method includes particles including a polysaccharide-based material and no metal carbonate, and particles including a metal carbonate and no polysaccharide-based material. In some embodiments, by "particles including only one of (i) a polysaccharide-based material or (ii) a metal carbonate" is meant that the polysaccharide-based material and the metal carbonate are not combined, processed together, bonded, linked, or tethered to each other, e.g., by processing and / or any chemical interaction. In some embodiments, the terms "particles comprising only one of (i) a polysaccharide-based material or (ii) a metal carbonate" and "each of the polysaccharide-based material and the metal carbonate in different particles" are interchangeable. In some embodiments, a particle characterized as a method or composition "comprising each of the polysaccharide-based material and the metal carbonate in different particles" means that the polysaccharide-based material and the metal carbonate are not included in the same particle, i.e., the polysaccharide-based material and the metal carbonate are not combined, processed together, bonded, linked, and tethered to each other, such as by processing and / or any chemical interaction. In such embodiments, the particle may comprise only one of (i) a polysaccharide-based material or (ii) a metal carbonate, and optionally additional materials. For example, the composition and / or method may include particles comprising a polysaccharide-based material and no metal carbonate and particles comprising a metal carbonate and no polysaccharide-based material. In some embodiments, the particles comprising the polysaccharide-based material and without the metal carbonate and the particles comprising the metal carbonate and without the polysaccharide-based material are suspended in solution in close proximity to one another during contact or incubation. In some embodiments, each particle type comprises at least one additional material. In some embodiments, the composition and / or method further comprises another type of particle.In some embodiments, the composition and / or method further comprises other particle types that lack both the polysaccharide-based material and the metal carbonate, such as particles comprising dicalcium phosphate (DCP). The other particle types can be blended particles or hybrid particles. In some embodiments, the composition and / or method comprises a plurality of different or identical composite particles, such as particles comprising the polysaccharide-based material and the metal carbonate and / or particles comprising dicalcium phosphate (DCP) and the polysaccharide-based material. In some embodiments, a particle consisting essentially of a metal carbonate is considered a composite particle.

[0085] In some embodiments, the metal carbonate particles are not produced by the at least one bacterial strain. In some embodiments, the composition and / or method includes a metal carbonate that has been produced by the at least one bacterial strain. In some embodiments, the metal carbonate is formed or produced prior to culturing the at least one bacterial strain. In some embodiments, the metal carbonate is a water-insoluble metal carbonate. In some embodiments, the metal is provided in a non-consumable form and / or is water-insoluble. In some embodiments, the metal carbonate is not produced by a bacterial biofilm. In some embodiments, the metal carbonate is not produced by a particle-attached bacterium of the invention. In some embodiments, the polysaccharide-based material is provided in a non-consumable form. In some embodiments, the polysaccharide-based material is not produced by the at least one bacterial strain, e.g., is not an autologous exopolysaccharide. In some embodiments, the composition and / or method includes a polysaccharide-based material that has been produced by the at least one bacterial strain. In some embodiments, the polysaccharide-based material is formed or produced prior to culturing the at least one bacterial strain. In some embodiments, the polysaccharide-based material is water insoluble. In some embodiments, the polysaccharide-based material is not produced by the particle-attached bacteria of the present invention. In some embodiments, at least one bacterial strain produces metal carbonates and / or the polysaccharide-based material. In some embodiments, the particles are not produced from media precipitates.

[0086] In some embodiments, the method for preparing the composition includes providing a population of at least one bacterial strain, providing particles comprising (i) a polysaccharide-based material and (ii) a metal carbonate, contacting the population of at least one bacterial strain with the particles, the contacting being performed in a solution, and at least partially attaching the population of at least one bacterial strain to the particles. In some embodiments, during the contacting or culturing step, the suspended particles have a characteristic selected from (a) being in the form of a composite comprising a polysaccharide-based material and a metal carbonate, (b) comprising only one of (i) the polysaccharide-based material or (ii) the metal carbonate, or both (a) and (b). In some embodiments, the particles comprising only one of (i) the polysaccharide-based material or (ii) the metal carbonate include particles comprising the polysaccharide-based material without the metal carbonate and particles comprising the metal carbonate without the polysaccharide-based material.

[0087] As used herein, the term "population of at least one bacterial strain" refers to any integer equal to or greater than one. In some embodiments, the composition comprises a population of one bacterial strain and / or the method comprises contacting or culturing a population of one bacterial strain. In some embodiments, the population of at least one strain comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 or more bacterial strains. In the latter embodiment, the bacteria may be from one or more taxonomic classifications.

[0088] In some embodiments, the population of at least one bacterial strain originates or is derived from a biological sample. In some embodiments, the population of at least one bacterial strain originates or is derived from a human body fluid, such as breast milk, vaginal fluid, etc. In some embodiments, the population of at least one bacterial strain is a synthetic sample. In some embodiments, the term "synthetic sample" refers to a sample that contains an artificially created bacterial community by combining / mixing selected (two or more) bacterial species.

[0089] As used herein, the terms "originating" and "derived" and their conjugations are interchangeable with the term "obtained" and refer to the source from which the bacteria for culture / fermentation are obtained.

[0090] In some embodiments, the population of at least one bacterial strain originates or is derived from a fecal sample, an oral sample (e.g., a saliva sample), a skin sample, an eye sample, a bronchial sample, a vaginal sample, or any combination thereof. In some embodiments, the population of at least one bacterial strain originates from a fecal bacterial population, a gut bacterial population, an eye bacterial population, an oral bacterial population (e.g., a saliva bacterial population), a skin bacterial population, a bronchial bacterial population, a vaginal bacterial population, an upper respiratory tract bacterial population, a urogenital tract bacterial population, or any combination thereof. In some embodiments, the population of at least one bacterial strain originates from a soil bacterial population, a groundwater bacterial population, an open water bacterial population, or any combination thereof. In some embodiments, the population of at least one bacterial strain originates or is derived from a plant. In some embodiments, the bacterial population originates or is derived from two or more bacterial sources.

[0091] In some embodiments, the population comprises at least one mutant strain. As used herein, the term "mutant strain" encompasses additions, deletions, substitutions, indels, inversions, duplications, or any combination thereof, for example, as compared to a naturally occurring bacterial strain (such as, but not limited to, a wild-type strain) or a genetic reference strain.

[0092] In some embodiments, at least one bacterial strain is a probiotic strain. In some embodiments, the term "probiotic" refers to a bacterial strain that, when administered in sufficient amounts, can provide a health benefit to a host (e.g., a human), such as ameliorating any disease, disorder, and / or condition in a subject, such as by improving or restoring healthy flora, affecting the metabolic processes of the subject, and / or stimulating the growth of other microorganisms, particularly microorganisms with beneficial properties.

[0093] In some embodiments, the at least one bacterial strain population is gram-positive, gram-negative, or both. In some embodiments, the at least one bacterial strain population comprises facultative anaerobes, resistant anaerobes, obligate anaerobes, or any combination thereof. In some embodiments, the at least one bacterial strain population comprises obligate aerobes, microaerobes, or any combination thereof. In some embodiments, the at least one bacterial strain population is isolated from a donor sample. In some embodiments, a single isolated colony is selected from a donor sample. In some embodiments, the entire sample is used in the method of the present invention, and the composition comprises a mixture of bacterial strains. In some embodiments, the at least one bacterial strain population comprises those originating from fecal microbiota, vaginal microbiota, for example, a collection of microorganisms colonizing a subject.

[0094] The bacterial strains can vary. In some embodiments, the population of at least one bacterial strain belongs to the bacterial family Lactobacillaceae, Bifidobacteriaceae. In some embodiments, the population of at least one bacterial strain belongs to the bacterial family Lactobacillaceae, Bifidobacteriaceae. 2 O 2 )-producing bacterial species. In some embodiments, the population of at least one bacterial strain is capable of colonizing epithelial-lined tissue.

[0095] In some embodiments, the population of at least one bacterial strain comprises a Lactobacillus strain. In some embodiments, the Lactobacillus strain is characterized by or has the ability to colonize epithelial-lined tissue. In some embodiments, the epithelial-lined tissue comprises vaginal tissue. In some embodiments, the population of at least one bacterial strain comprises at least one of the following species: Lactobacillus iners, Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus vaginalis, Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus helveticus, Lactobacillus helveticus, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus salivarius, Lactobacillus delbrueckii, or any combination thereof. In some embodiments, the population of at least one bacterial strain comprises at least one bacterium selected from Lactobacillus clipatus, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus rhamnosus, and any combination thereof. In some embodiments, the population of at least one bacterial strain comprises Lactobacillus clipatus. In some embodiments, the population of at least one bacterial strain comprises Lactobacillus rhamnosus.In some embodiments, the population of at least one bacterial strain comprises Lactobacillus paracasei.

[0096] In some embodiments, the population of at least one bacterial strain comprises at least one bacterial strain characterized by or equipped with the ability to colonize skin tissue. In some embodiments, the population of at least one bacterial strain comprises at least one of the following species: Lactobacillus plantarum, Propionibacterium acnes, Corynebacterium tuberculostearicum, Propionibacterium acnes, Corynebacterium tuberculostearicum, Corynebacterium tuberculostearicum, Staphylococcus hominis, Staphylococcus epidermidis, Staphylococcus hominis, Streptococcus mitis, Propionibacterium acnes, Corynebacterium tuberculostearicum, Staphylococcus warneri, Staphylococcus warneri, Streptococcus oralis, Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus epidermidis, Streptococcus pseudopneumoniae, Staphylococcus capitis, Corynebacterium simulans, Staphylococcus capitis, Streptococcus sanguinis, Corynebacterium fastidiosum, Streptococcus mitis, Staphylococcus haemolyticus, Micrococcus luteus luteus, Corynebacterium afermentansafermentans, Staphylococcus hominis, Micrococcus luteus, Staphylococcus epidermidis, Micrococcus luteus, Corynebacterium aurimucosum, Corynebacterium afermentans, Staphylococcus capitis, Enhydrobacter aerosaccus, Corynebacterium kroppenstedtii, Corynebacterium simulans, Veillonella parvula, Corynebacterium simulans, Corynebacterium amycolatum, Corynebacterium resistens resistens, Lactobacillus pentosus, Lactobacillus rhamnosus, or any combination thereof. In some embodiments, the population of at least one bacterial strain comprises Lactobacillus plantarum.

[0097] In some embodiments, the population of at least one bacterial strain comprises a Staphylococcus strain, such as Staphylococcus epidermidis. In some embodiments, the population of at least one bacterial strain comprises a Bifidobacterium strain, such as Bifidobacterium bifidum. In some embodiments, the population of at least one bacterial strain comprises Escherichia coli Nissle 1917.

[0098] In some embodiments, the phrase "bacteria at least partially attached to particles" refers to 0.01%-100%, 1%-95%, 5%-90%, or 10%-85% of the total bacteria present in the composition and / or at the end of the contacting or culturing step, such as at least 0.01%, at least 0.05%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%, or any value and range therebetween, being in a form attached / adhered to the particles. In some embodiments, the bacteria are attached / adhered to at least one of (i) the surface of the polysaccharide-based material, (ii) the surface of the metal carbonate material, or to both (i) and (ii). In some embodiments, the majority of the attached bacteria are attached to the surface of the metal carbonate particles (e.g., greater than 50% up to 100% of the total attached bacteria fraction, e.g., at least 51%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or even 100%, or any values ​​and ranges therebetween).

[0099] In some embodiments, "bacteria at least partially attached to a particle" includes direct and indirect attachment or adhesion of bacteria to a particle. In some embodiments, indirect attachment includes attachment to adherent bacteria and / or attachment to a matrix formed by the adherent / adherent bacteria.

[0100] In some embodiments, adhesion can be determined by scanning electron microscopy (SEM) techniques, and / or by separating the adherent and non-adherent bacterial fractions, e.g., by filtration and / or gravity settling, e.g., centrifugation, and determining the viability of the bacteria in the adherent fraction, and / or by any other method known to those skilled in the art. The total bacteria present in the composition and / or the total bacteria present in both the adherent and non-adherent fractions at the end of the contacting or culturing step can be determined by any method known to those skilled in the art for bacterial viability, e.g., the methods described herein. In some embodiments, the bacteria are separated from the particles prior to determining the viability of the bacteria.

[0101] In some embodiments, bacterial adhesion and the percentage of bacterial adhesion among all bacteria present in the culture system are evaluated as follows: The particle-attached bacterial fraction is separated from the non-attached fraction by centrifugation (e.g., 48×g for 3 min at 21±2° C.) and removing the supernatant containing the non-attached bacteria. In some embodiments, the pellet of the particle-attached bacterial fraction is obtained by washing the pellet with 0.9% saline, repeating the centrifugation (e.g., 48×g for 3 min at 21±2° C.) and removing the supernatant. The pellet is resuspended in 0.9% saline and a drop assay can be performed on the resuspended fraction to evaluate bacterial viability in the attached fraction. The percentage of bacterial adhesion can be evaluated by determining the bacterial viability of the whole sample before separating the whole sample into two fractions and calculating the bacterial viability in the attached fraction of the viability of the whole sample.

[0102] In some embodiments, the term "particle" refers to a substance / material adapted, configured, or suitable for the attachment and / or growth of at least one bacterial strain. In some embodiments, the particle comprises a surface to which bacteria can adhere. In some embodiments, the term "surface" in the context of a particle is interchangeable with the term "surface area" and refers to the outer surface of the particle. In some embodiments, the particle is porous. In some embodiments, the term "surface" includes any outer surface of the porous structure of the particle, including the internal voids of the particle. In some embodiments, the particle is non-porous. In some embodiments, the particle is a mixture of porous and non-porous particles. In some embodiments, the term "porous" refers to void (i.e., "empty") space in a material. In some embodiments, the term "porous" includes having an irregular surface area. In some embodiments, the term "particle" refers to a material with a discontinuous three-dimensional shape that is separate and separable from other substances / components added in the method, although this does not exclude the components from contacting each other.

[0103] In some embodiments, when the population of at least one bacterial strain originates or is derived from a biological sample, the particles do not originate from the sample. In some embodiments, when the population of at least one bacterial strain originates or is derived from a biological sample, the sample lacks the particles disclosed herein.

[0104] As used herein, the terms "particle," "nanoparticle," "microparticle," "nanosphere," and "microsphere" are used interchangeably.

[0105] In some embodiments, all numerical percentages and w / w ratios herein for particles are based on the particles in dry form. In some embodiments, the term "dry" for particle percentages and weight characteristics is interchangeable with the term "non-hydrated". In some embodiments, the term "dry" refers to particles that have been subjected to a drying process. In some embodiments, the term "dry" refers to a moisture content in the particle that is less than 5%. In some embodiments, "dry particles" refers to particles that do not contain unbound water. In some embodiments, the particles contain bound water. In some embodiments, where the particles are prepared by precipitation into a culture medium, the dry weight can be calculated by isolating the particles from the culture medium, for example by centrifugation, removing the liquid phase, and subjecting the isolated particles to a drying step until a moisture content of less than 5% is obtained. In some embodiments, the isolated particles are first subjected to a step of separating the attached bacteria, for example by vortexing. The moisture content can be determined by Karl Fischer titration, loss on drying (LOD), and / or any other method known to one skilled in the art.

[0106] In some embodiments, the particles are water-insoluble particles. In some embodiments, the particles include water-insoluble particles. In some embodiments, the term "water-insoluble" refers to particles that are not substantially soluble in water, such as saline, phosphate buffered saline, and growth media. In some embodiments, the term "substantially insoluble" means that at least 90 wt.% of the particles, preferably 95 wt.% of the particles, are not soluble in water. In some embodiments, the particles have a water solubility of 2.5 g / 1,000 ml or less at a temperature in the range of 20° C. to 60° C., such as a temperature selected from 20, 25, 37, 60° C., and / or at a pH in the range of 5.0 to 8.0. Each possibility represents a separate embodiment of the invention. In some embodiments, the particles used in accordance with the invention are water-insoluble at physiological pH. In some embodiments, the particles used in accordance with the invention are water-insoluble at a pH in the range of about 1.0 to 8.0.

[0107] In some embodiments, the compositions and / or methods include multiple types of particles, e.g., having different size distributions. In some embodiments, the particles range from 0.1 microns to 1 cm in diameter. In some embodiments, the particles range from 5 microns to 1 cm in diameter. In some embodiments, the particles range from 0.1 microns to 500 microns in diameter. In some embodiments, the particles range from 1 micron to 50 millimeters in diameter. In some embodiments, the compositions and / or methods include particles smaller than 1 micron in diameter. In some embodiments, the compositions and / or methods include particles in the range of 30-500 microns (e.g., 40-400 microns, 50-300 microns, 60-200 microns, 70-100 microns, or any values ​​and ranges therebetween). In some embodiments, the compositions and / or methods include particles in the range of 0.2 to 100 microns (e.g., 1 to 90 microns, 1 to 80 microns, 1 to 70 microns, 1 to 60 microns, 1 to 50 microns, 1 to 40 microns, 1 to 30 microns, 1 to 25 microns, 1 to 20 microns, 2 to 90 microns, 4 to 80 microns, 6 to 70 microns, 8 to 60 microns, 10 to 50 microns, 12 to 40 microns, 14 to 30 microns, 16 to 20 microns, or any value and range therebetween), or any value and range therebetween.In some embodiments, the particles are at least 0.2 microns in diameter, at least 2 microns in diameter, at least 3 microns in diameter, at least 5 microns in diameter, at least 10 microns in diameter, at least 15 microns in diameter, at least 20 microns in diameter, at least 30 microns in diameter, at least 40 microns in diameter, at least 50 microns in diameter, at least 60 microns in diameter, at least 70 microns in diameter, at least 80 microns in diameter, at least 90 microns in diameter, at least 100 microns in diameter, at least 200 microns in diameter, at least at least 300 microns in diameter, at least 400 microns in diameter, at least 500 microns in diameter, at least 600 microns in diameter, at least 700 microns in diameter, at least 800 microns in diameter, at least 900 microns in diameter, at least 1 cm in diameter, or any values ​​and ranges therebetween. Each possibility represents a separate embodiment of the present invention.

[0108] In some embodiments, the average diameter of the particles is in the range of 0.2 to 1,500 microns. In some embodiments, the average diameter of the particles is in the range of 50 to 1,200 microns, 50 to 1,100 microns, 50 to 1,000 microns, 55 to 1,200 microns, 55 to 1,000 microns, 57 to 1,200 microns, or 60 to 1000 microns (including any range therebetween). Each possibility represents a separate embodiment of the present invention.

[0109] In some embodiments, the diameter is an average diameter. In some embodiments, the diameter is a maximum diameter. In some embodiments, the diameter is a minimum diameter.

[0110] In some embodiments, the term "particle" includes any particle, e.g., in the specifically exemplified size ranges, of any shape, in some embodiments, the particles can be round, amorphous, irregular, spherical, elliptical, flower, cubic, spherical, elongated, rod-like, have any other shape, or any combination thereof.

[0111] In some embodiments, at least a portion of the particles comprise a composite or mixture comprising (i) the polysaccharide-based material and (ii) the metal carbonate. In some embodiments, the particles are in the form of a composite comprising the polysaccharide-based material and the metal carbonate during the contacting and / or culturing steps. In some embodiments, the composite particles are formed during the contacting and / or culturing steps, such as by chemical interaction. In some embodiments, the particles are provided to the method as a composite or mixture comprising (i) the polysaccharide-based material and (ii) the metal carbonate.

[0112] In some embodiments, the term "at least a portion of the particles comprise a composite or mixture comprising (i) a polysaccharide-based material and (ii) a metal carbonate" refers to a range of 1%-100%, 5%-90%, 10%-80%, or 20%-70%, such as at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or even 100% (by weight), of the particles being in the form of a composite particle comprising both the polysaccharide-based material and the metal carbonate, or any value and range therebetween. All percentages are percentages of the total particles present in the composition and / or provided, added, or used in the method. In some embodiments, the remaining particles are in a form that includes only one of the polysaccharide-based material or the metal carbonate. In some embodiments, the "composite particles" and / or the "remaining particles" include another type of material.

[0113] As used herein, the terms "composite" or "mixture" refer to particles made up of different materials. In some embodiments, the composite particles include (i) a polysaccharide-based material and (ii) a metal carbonate, and optionally additional materials or substances.

[0114] In some embodiments, a portion of the particles comprises only one of the polysaccharide-based material or the metal carbonate. In some embodiments, the term "portion" with respect to particles comprising only one of the polysaccharide-based material or the metal carbonate refers to the remaining percentage of the particles in the composition and / or particles provided, added, or used in the method.

[0115] In some embodiments, the particles are separate particles comprising only one of the polysaccharide-based material or the metal carbonate during the contacting and / or culturing steps. In some embodiments, the polysaccharide-based material and the metal carbonate are provided, added, or used in the method as separate particles each comprising only one of the polysaccharide-based material or the metal carbonate. In some embodiments, the term "separate" includes the terms "individual," "separate," "different," or "uncombined" and refers to the composition of the particles for the polysaccharide-based material and the metal carbonate. In some embodiments, the "separate particles" are combined or mixed with additional materials. In some embodiments, the separate particles form a composite during and / or after contact with the solution.

[0116] In some embodiments, the particles in the composition and / or particles provided, added, or used in the method that comprise only one of the polysaccharide-based material or the metal carbonate are less than 100%, such as less than 99%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% (by weight), or any values ​​and ranges therebetween. All percentages are percentages of the total particles in the composition and / or the total particles provided, added, or used in the method.

[0117] In some embodiments, a majority of the particles in the composition and / or particles provided, added or used in the method (e.g., greater than 50%, e.g., 51%-100%, at least 51%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or even 100% (by weight), or any values ​​and ranges therebetween) are composite particles comprising at least a combination of a polysaccharide-based material and a metal carbonate. All percentages are percentages of the total particles in the composition and / or method.

[0118] In some embodiments, the compositions and / or methods include multiple particle types, such as those that include different materials. In some embodiments, the compositions and / or methods include different types of polysaccharide-based materials, different types of metal carbonates, or any combination thereof. In some embodiments, each particle can include a different type of polysaccharide-based material and / or a different type of metal carbonate.

[0119] In some embodiments, each particle includes an additional material, thereby forming a "combined or hybrid particle." In some embodiments, the additional material includes dicalcium phosphate (DCP). In some embodiments, each particle includes one material. In some embodiments, each particle includes two or more different materials. In some embodiments, the composition and / or method further includes particles that lack a polysaccharide-based material and a metal carbonate. In some embodiments, the composition and / or method includes particles that include DCP, a polysaccharide-based material, or a combination thereof.

[0120] In some embodiments, the material is homogeneously distributed within an individual particle. The term "homogeneous" means that the material is substantially evenly distributed throughout an individual particle, e.g., without being present in a substantially higher concentration in any one portion of the particle. In some embodiments, different regions of a particle have approximately the same material composition. In some embodiments, different regions of a particle have different material compositions.

[0121] In some embodiments, the composite particles comprising a polysaccharide-based material and a metal carbonate have at least one characteristic selected from the following: (i) have not been subjected to a physical treatment, (ii) have been subjected to a physical treatment, (iii) are not a co-processed mixture, (iv) are a co-processed mixture, and (v) the polysaccharide-based material and the metal carbonate are bonded, linked, or tethered by a chemical interaction.

[0122] In some embodiments, the compositions and / or methods include blended or hybrid particles, e.g., those comprising a mixture of various materials, that have at least one characteristic selected from the following: (i) not having been subjected to physical treatment, (ii) having been subjected to physical treatment, (iii) not being a co-processed mixture, (iv) being a co-processed mixture, and (v) the materials that make up the particle are bonded, linked, or connected by chemical interactions.

[0123] In some embodiments, the compositions and / or methods include a plurality of composite, blended, or hybrid particles comprising any combination of features (i)-(v).

[0124] In some embodiments, physical processing includes compression, shear stress, etc. In some embodiments, particles in the form of a co-processed mixture include materials / substances that are combined by applying mechanical pressure.

[0125] In some embodiments, the composite, blended or hybrid particles are added as separate materials in the method, such as by any chemical bonds, forces and / or interactions, to form the composite, blended or hybrid particles.

[0126] In some embodiments, the method further comprises dispersing the polysaccharide-based material and the metal carbonate in a solution. In some embodiments, the polysaccharide-based material and the metal carbonate are dispersed together in the same solution or in separate solutions followed by a step of combining the two dispersions. In some embodiments, the dispersing step is performed simultaneously, sequentially and / or alternatingly in the same solution. In some embodiments, the method further comprises mixing one or more solutions containing the dispersed polysaccharide-based material and the metal carbonate, e.g., by simple blending. In some embodiments, simple blending includes conventional mixing or blending operations, e.g., shaking, stirring, etc. In some embodiments, composite particles containing the polysaccharide-based material and the metal carbonate are formed by dispersing the two separate particle types in one solution and / or by mixing / combining two separate dispersion solutions, each containing a different particle type / material. Non-limiting examples of solutions are saline, phosphate buffer, growth medium, etc. In some embodiments, the dispersing is performed before, simultaneously with, and / or after inoculating or adding the bacteria to the solution. In some embodiments, the solution after combination is a homogenous mixture of dispersed particle types. In some embodiments, one or more additional particle types are dispersed in the solution. In some embodiments, the polysaccharide-based material and the metal carbonate, and optionally the additional particle types, are dispersed in the solution simultaneously, sequentially, or both. In some embodiments, the particles / materials are dispersed in the solution in dry form, e.g., in powder form, in wet form, or both.

[0127] In some embodiments, at least a portion of the particles are blended, hybrid, or composite particles comprising a mixture of two or more substances. In some embodiments, a portion of the particles comprises a mixture of a polysaccharide-based material and a metal carbonate. In some embodiments, the composition and / or method comprises a particle comprising a polysaccharide-based material. In some embodiments, the composition and / or method comprises a particle comprising a metal carbonate. In some embodiments, the composition and / or method comprises a particle comprising only one of a polysaccharide-based material or a metal carbonate, and optionally an additional material. In some embodiments, the composition and / or method comprises a particle comprising a polysaccharide-based material and substantially free of a metal carbonate. In some embodiments, the composition and / or method further comprises a particle comprising a metal carbonate and substantially free of a polysaccharide-based material. In some embodiments, the composition and / or method comprises a particle consisting essentially of (i) a polysaccharide-based material and a metal carbonate, (ii) a polysaccharide-based material, (iii) a metal carbonate, or (iv) any combination of (i)-(iii).

[0128] In some embodiments, the weight ratio between (i) particles comprising polysaccharide-based material and substantially free of metal carbonate, (ii) particles comprising metal carbonate and substantially free of polysaccharide-based material, and (iii) composite particles comprising polysaccharide-based material and metal carbonate ranges from 1:1:1 to 10:10:10, such as 1:1:10, 10:1:1, 1:10:1, 1:10:10, 10:10:1, 10:1:10, or any value and range therebetween. All ratios are based on a weight / weight / weight (w / w / w) basis of the dry particle form.

[0129] In some embodiments, "polysaccharide-based material" refers to a material that includes polysaccharides, whereas the term "polysaccharide" refers to a pure carbohydrate, such as starch or cellulose. In some embodiments, "polysaccharide-based material" refers to a natural polymer composed of glucose units linked by, for example, 1-4 β-glycosidic bonds. In some embodiments, a polysaccharide-based material is a material that forms linear polysaccharides, e.g., crystalline or partially crystalline structures, at temperatures, e.g., in the range of 20-60° C., e.g., at a temperature selected from 20, 25, 37, 60° C., and / or at a pH in the range of 5-8. In some embodiments, the term "polysaccharide-based material" includes oligosaccharide-based materials.

[0130] In some embodiments, the polysaccharide-based material includes materials / substances based on cellulose, pectin, inulin, dextrin, maltodextrin, glycogen, starch, such as those derived from corn or potato, amylose, alginate, chitosan, such as hydrophobically modified chitosan (HMC), fructooligosaccharides (FOS), galactooligosaccharides (GOS), or any combination thereof.

[0131] In some embodiments, the polysaccharide-based material comprises fructooligosaccharides (FOS), galactooligosaccharides (GOS), or a combination thereof. In some embodiments, the cellulosic material comprises a material that includes cellulose fibers, such as microcrystalline cellulose (MCC).

[0132] In some embodiments, the polysaccharide-based material is characterized by having a positive net charge, a negative net charge, or a neutral net charge. In some embodiments, the polysaccharide-based material comprises a surface having a charge. As used herein, the term "net charge" refers to the overall charge of the material. For example, a material with a positive net charge is a material that has an overall excess of protons. In embodiments where the composition and / or method includes multiple types of polysaccharide-based materials, each type of polysaccharide-based material may have a different net charge. In some embodiments, the multiple types of polysaccharide-based materials are characterized by having a positive net charge, a negative net charge, a neutral net charge, or any combination thereof.

[0133] In some embodiments, the polysaccharide-based material is negatively charged, for example, MCC is negatively charged at a pH range of 3.5 to 10.0.

[0134] In some embodiments, the term "metal carbonate" is interchangeable with the term "mineral salt" and refers to a metal cation and a carbonate anion (CO 3 -2 ). Typically, metal carbonates undergo decomposition, for example when reacted with an acid, to produce salt, water, and carbon dioxide gas (CO 2 ) can be generated.

[0135] In some embodiments, the metal ions / positively charged metal ions / metal cations are in the form of monovalent, divalent, and trivalent metal ions. In some embodiments, the compositions and / or methods include multiple types of metal carbonates that include multiple types of metal cations. In some embodiments, the metal cations include monovalent, divalent, trivalent metal cations, or any combination thereof.

[0136] In some embodiments, the metal carbonate is or comprises a divalent cation. In some embodiments, the metal cation is calcium (Ca 2+ ), Barium (Ba 2+ ), Manganese (Mn 2+ ), iron (Fe2+ ), Magnesium (Mg 2+ ), Zinc (Zn 2+ ), or any combination thereof. In some embodiments, the metal cation is or includes calcium (Ca 2+ ).

[0137] In some embodiments, the metal cation is a calcium cation (Ca 2+ ), and the metal carbonate is calcium carbonate (CaCO 3 In some embodiments, the polysaccharide-based material is or comprises a cellulose derivative, such as microcrystalline cellulose (MCC). In some embodiments, at least a portion of the particles in the compositions and / or methods are in the form of composite particles comprising a mixture of (i) MCC and (ii) calcium carbonate. In some embodiments, at least a portion of the particles in the compositions and / or methods comprise (i) MCC and (ii) calcium carbonate as separate, uncombined particles. In some embodiments, each particle further comprises an additional material.

[0138] "Calcium carbonate" (CaCO) that can be used in accordance with the present invention 3 ) can be synthesized or produced by any method known in the art, for example to obtain nanometer and micrometer / micron materials, and are typically available in several forms, shapes and crystal structures. In some embodiments, the forms and types used in accordance with the present invention are suitable for human consumption. In some embodiments, the calcium carbonate is derived from natural sources, synthetic sources, or a combination thereof.

[0139] In some embodiments, the metal carbonate-containing particles, e.g., calcium carbonate, are precipitated particles. In some embodiments, the metal carbonate used in accordance with the present invention is a pre-made particle, e.g., commercially available. In some embodiments, the precipitated particles are formed in a vessel used to contact the bacteria with the particles. In some embodiments, the method further comprises precipitating (e.g., dropping) calcium oxide into a solution (e.g., contained within a culture vessel), thereby providing particles comprising calcium carbonate. In some embodiments, the method further comprises drying Na 2 CO 3 and CaCl 2 and precipitated (e.g., dropped) into solution and / or 2 CO 3 and CaCl 2 and mixing the solution of the calcium carbonate particles with the calcium carbonate-containing particles to thereby provide particles comprising calcium carbonate. In some embodiments, the solution is water, saline, phosphate buffered saline, or growth medium. In some embodiments, the calcium carbonate particles are purified, refined, and / or synthetic calcium carbonate. In some embodiments, the calcium carbonate-containing particles are ground calcium carbonate. In some embodiments, the calcium carbonate comprises impurities.

[0140] In some embodiments, the average diameter of the calcium carbonate particles is in the range of 0.2 to 100 microns, such as 2 to 20 microns, 2.1 to 3 microns, 0.2 to 5 microns, 20 to 100 microns, or any value and range therebetween. In some embodiments, the calcium carbonate is amorphous CaCO 3 , crystalline CaCO 3 In some embodiments, the calcium carbonate is calcite (β-CaCO 3 ), aragonite (λ-CaCO 3 ), vaterite (μ-CaCO 3 ), and any combination thereof.

[0141] In some embodiments, during contacting and / or culturing, the metal carbonate and polysaccharide-based material are present at concentrations that allow for at least partial attachment of the population to the particles.

[0142] Without being bound to a particular theory or mechanism, the use of high metal carbonate concentrations results in a highly viscous culture medium, which may require increased stirring speed to obtain a uniform culture medium, thus adversely affecting bacterial adhesion to the particles, while the use of low metal carbonate concentrations also results in less bacterial adhesion.

[0143] Thus, in some embodiments, the total weight of particles relative to the volume of solution, e.g., growth medium, during the contacting and / or culturing step is up to 250 g per liter. In some embodiments, the total weight of particles relative to the volume of solution, e.g., growth medium, during the contacting and / or culturing step is at least 60 g per liter. In some embodiments, the total weight of particles relative to the volume of solution, e.g., growth medium, during the contacting and / or culturing step is in the range of 60 g per liter to 250 g per liter, e.g., 60 g per liter to 200 g per liter.

[0144] In some embodiments, at such particle:solution ratios, the metal carbonate comprises 85% w / w or less of the total weight of the particle. In some embodiments, at such particle:solution ratios, the metal carbonate comprises up to 85% w / w of the total weight of the particle. In some embodiments, at such particle:solution ratios, the metal carbonate comprises up to 75% w / w of the total weight of the particle. In some embodiments, at such particle:solution ratios, the metal carbonate comprises 75% w / w or less of the total weight of the particle. In some embodiments, at such particle:solution ratios, the metal carbonate comprises at least 15% w / w of the total weight of the particle. In some embodiments, at such particle:solution ratios, the metal carbonate comprises at least 25% w / w of the total weight of the particle. In some embodiments, at such particle:solution ratios, the metal carbonate content ranges from 15% to 85% w / w of the total weight of the particle. In some embodiments, at such particle:solution ratios, the metal carbonate content ranges from 25% to 75% w / w of the total weight of the particle. All percentages are w / w relative to the total weight of the particles provided, added, or used in the method. Thus, in some embodiments, the metal carbonate comprises at least 15% w / w of the total weight of the particles in the composition. In some embodiments, the metal carbonate comprises at least 25% w / w of the total weight of the particles in the composition. In some embodiments, the metal carbonate comprises up to 85% w / w of the total weight of the particles in the composition. In some embodiments, the metal carbonate is 85% w / w or less of the total weight of the particles in the composition. In some embodiments, the metal carbonate comprises up to 75% w / w of the total weight of the particles in the composition. In some embodiments, the metal carbonate is 75% w / w or less of the total weight of the particles in the composition. In some embodiments, the metal carbonate content ranges from 25% to 75% w / w of the total weight of the particles in the composition. In some embodiments, the metal carbonate content ranges from 15% to 85% w / w of the total weight of the particles in the composition.

[0145] In some embodiments, the metal carbonate content in the composition and / or in the particles provided, added, or used in the method is between 15% and 85% (w / w), between 15% and 80% (w / w), between 15% and 75% (w / w), between 15% and 70% (w / w), between 15% and 65% (w / w) of the total weight of the particles in the composition and / or the particles provided, added, or used in the method. , 15%-60%(w / w), 15%-55%(w / w), 15%-50%(w / w), 15%-45%(w / w), 15%-40%(w / w), 20%-85%(w / w), 25%-75%(w / w), 25%-85%(w / w), 25%-80%(w / w), 20%-50%(w / w), 20%-48%(w / w), 20%-45%(w / w), or 20%-40%(w / w).

[0146] In some embodiments, the remaining percentage is particles that include polysaccharide-based material. In some embodiments, the polysaccharide-based material content is 15%-85% (w / w), 15%-80% (w / w), 15%-75% (w / w), 15%-70% (w / w), 15%-65% (w / w), 15%-60% (w / w), 15%-55% of the total weight of the particles in the composition and / or particles provided, added, or used in the method. (w / w), 15%-50%(w / w), 15%-45%(w / w), 15%-40%(w / w), 20%-85%(w / w), 25%-75%(w / w), 25%-85%(w / w), 25%-80%(w / w), 20%-50%(w / w), 20%-48%(w / w), 20%-45%(w / w), or 20%-40%(w / w).

[0147] In some embodiments, the weight of the metal carbonate particles provided relative to the volume of solution is at least 9 g per liter. In some embodiments, the weight of the metal carbonate particles provided relative to the volume of solution is up to 215 g per liter. In some embodiments, the weight of the metal carbonate particles provided relative to the volume of solution is in the range of 9 g per liter to 215 g per liter, 9 g per liter to 200 g per liter, 15 g per liter to 190 g per liter.

[0148] In some embodiments, the weight of the polysaccharide-based particles provided relative to the volume of solution is at least 9 g per liter. In some embodiments, the weight of the polysaccharide-based particles provided relative to the volume of solution is up to 215 g per liter. In some embodiments, the weight of the polysaccharide-based particles provided relative to the volume of solution is in the range of 9 g per liter to 215 g per liter, 9 g per liter to 200 g per liter, 15 g per liter to 190 g per liter.

[0149] In some embodiments, the polysaccharide-based material, e.g., MCC, and the metal carbonate, e.g., calcium carbonate, are present in the composition and / or solution during the contacting or culturing step in a weight / weight (w / w) ratio of 1:10: to 10:1, 1:6 to 6:1, or any value and range therebetween. In some embodiments, the polysaccharide-based material and the metal carbonate are present in a weight / weight (w / w) ratio of 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0150] In some embodiments, the particles further comprise a metal phosphate. In some embodiments, the composite particles further comprise a metal phosphate. In some embodiments, the metal phosphate comprises dicalcium phosphate (DCP). In some embodiments, "metal phosphate" refers to an inorganic compound comprising a metal cation and phosphoric acid.

[0151] In some embodiments, when the particle further comprises a metal phosphate, the metal phosphate and metal carbonate collectively comprise at least 15% (w / w) of the total weight of the particle. In some embodiments, the ratio between the metal phosphate and metal carbonate ranges from 1:14 to 14:1, such as 2:13, 4:11, 6:9, 8:7, 13:2, 11:4, 9:6, 7:8, 2:13, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, when the particle further comprises a metal phosphate, the metal phosphate and metal carbonate collectively comprise at least 25% (w / w) of the total weight of the particle. In some embodiments, the ratio between the metal phosphate and metal carbonate ranges from 1:24 to 24:1, such as 7:18, 9:16, 11:14, 13:12, 18:7, 16:9, 14:11, 12:13, 7:18, or any value and range therebetween, with each possibility representing a separate embodiment of the present invention.

[0152] In some embodiments, during the contacting and / or culturing step, when the total weight of the particles relative to the volume of the solution, e.g., growth medium, is less than 60 g per liter, the metal carbonate concentration can be greater than 85% w / w of the total weight of the particles. In such embodiments, the metal carbonate concentration can be up to 99% w / w of the total weight of the particles. In another embodiment, the metal carbonate concentration is at least 15% w / w of the total weight of the particles. In another embodiment, the metal carbonate concentration is at least 25% w / w of the total weight of the particles. In some embodiments, the metal carbonate concentration is 15%-99% w / w of the total weight of the particles, e.g., 15%-95%, 15%-90%, 20%-95%, 25%-99%, 25%-90%, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. All percentages are w / w of the total weight of the particles provided, added, or used in the method.

[0153] Thus, in some embodiments, the metal carbonate comprises at least 15% w / w of the total weight of the particles in the composition. In some embodiments, the metal carbonate comprises at least 25% w / w of the total weight of the particles in the composition. In some embodiments, the metal carbonate comprises up to 99% w / w of the total weight of the particles in the composition. In some embodiments, the metal carbonate content ranges from 15%-99% of the total weight of the particles in the composition, such as 15%-95%, 15%-90%, 20%-95%, 25%-99%, 25%-90%, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0154] In some embodiments, CaCO 3 is at least partially entrapped and / or adsorbed by the polysaccharide-based material.

[0155] In some embodiments, a metal carbonate, such as CaCO 3 and the polysaccharide-based material are linked, adsorbed, fused, or superficially tethered to one another, for example, by any chemical bonds, forces and / or interactions.

[0156] In some embodiments, the method comprises contacting a population of at least one bacterial strain with the particles and at least partially attaching the population of at least one bacterial strain to the particles.

[0157] The term "contacting" is used herein in its broadest sense and refers to any type of mixing action, such as bringing bacteria into close proximity with particles so that the bacteria can attach / adhere to the particles. In some embodiments, contacting includes incubating, growing or culturing the bacteria with or in the presence of particles, as opposed to suspension culture, which is performed in the absence of particles. In some embodiments, contacting includes combining the bacteria, particles and solution in any order, any combination and / or subcombination, including any premixing of two components before adding a third component, or mixing all components simultaneously. In some embodiments, the method includes inoculating the solution with bacteria and then culturing the bacteria in the solution containing particles. In some embodiments, the method includes providing a solution containing particles as a preceding step, followed by adding a population of at least one bacterial strain. In some embodiments, contacting and culturing are performed simultaneously or as subsequent steps.

[0158] In some embodiments, the contacting or culturing is performed in a solution. In some embodiments, the solution is selected from the group consisting of a buffer, saline, phosphate buffered saline, and growth medium. In some embodiments, the contacting comprises culturing the bacteria in the presence of the particles.

[0159] In some embodiments, the term "cultivating" encompasses the term "fermentation" and refers to the in vitro maintenance, propagation and / or growth of cells, e.g., bacterial cells, in various types of buffers and / or media, under laboratory or industrial conditions. In some embodiments, the propagated or grown cells may not be completely identical in morphology, genetics or phenotype to the parent cells. The term "fermentation" has its normal meaning in the art. In some embodiments, the term "fermentation" is used herein to refer to a microbial metabolic process involving the conversion of sugars to acids and / or gases, such as by bacteria.

[0160] Suitable media will be apparent to those of skill in the art and may be any known or commercially available medium, or may be synthesized and / or custom designed for such purposes as to support the maintenance, proliferation and / or growth of cultured cells. Non-limiting examples of media include Tryptic Soy Broth (TSB), Yeast Casitone Fatty Acids (YCFA), Robertson's Cooked Meat, Reinforced Clostridial Broth (RCM) Broth, Nutrient Broth, Fastidious Anaerobic Broth (FAB), Lysogeny Broth (LB), 1 / 4 LB, Heart Infusion Broth, Wilkins-Chalgren broth, Brucella Broth, Wheat Bran, Brain Heart Infusion (BHI), Brain Heart Infusion Agar (BHI1), M9 Minimal Medium or 0.2x BHI, Gifu Anaerobic Broth (GAM), Gut Microbiota Medium (GCM), and / or 0.2x BHI. Medium (GMM), Columbia blood agar (CBA), chocolate agar (CHOC), tryptic soy agar (TSY), fastidious anaerobe agar (FAA), tryptic yeast extract glucose (TYG), cooked meat agar (BEEF), bifidobacterium selective medium (BSM), phenylethyl alcohol agar (PEA), actinomyces isolation agar (AIA), colistin naladixic acid agar (CNA), McKay agar (MK), mannitol slat agar (MSA), de Man Rogosa These include, but are not limited to, Sharpe agar (MRS), Bacteroides bile esculin agar (BBE), deoxycholate agar (DOC), Rogosa agar, CDC anaerobic blood agar (CDC), MacConkey agar (MAC), Staphylococcus broth, Bifidobacterium broth and Kanamycin-vancomycin hemolysate blood agar (KVLB), or any combination thereof and their equivalents.In some embodiments, the medium comprises pharma-grade components. In some embodiments, the medium comprises food-grade components. In some embodiments, the medium comprises components suitable for veterinary use.

[0161] In some embodiments, the method includes providing the bacteria contained in a vessel, and the contacting or culturing occurs in the vessel. In some embodiments, the term "vessel" refers to any receptacle in which the bacteria can be cultured by conventional fermentation techniques, such as a bioreactor, flask, test tube, microtiter dish, well plate, multi-well plate assembly, Petri plate, etc.

[0162] In some embodiments, the method further comprises inoculating the solution with a population of at least one bacterial strain, e.g., as a precursor to the contacting step. In some embodiments, the contacting comprises inoculating the solution with a population of at least one bacterial strain. In some embodiments, the contacting comprises incubating the population of at least one bacterial strain in a solution containing particles for a sufficient time for the at least one bacterial strain to at least partially adhere to the particles. In some embodiments, the contacting comprises culturing the population of at least one bacterial strain in, e.g., a growth medium. In some embodiments, the contacting comprises inoculating the solution containing particles with bacteria, and incubating or culturing the bacteria with the particles for a sufficient time for the bacteria to at least partially adhere to the particles.

[0163] In some embodiments, the contacting or culturing is performed under conditions that allow the population of at least one bacterial strain to at least partially attach to the particles. In some embodiments, suitable conditions include, for example, pH levels ranging from 5 to 8, flow, shaking, stirring, agitation, static, moist, low humidity, or any combination thereof. In some embodiments, suitable conditions include selecting a medium that supports the growth of the cultured bacterial strain. In some embodiments, suitable conditions are contacting or culturing the population of at least one bacterial strain with the particles for a period ranging from 1 hour to 15 days. In some embodiments, the time sufficient to allow the bacteria to at least partially attach / adhere to the particles ranges from 2 hours to 10 days, 2 hours to 8 days, 2 hours to 6 days, 2 hours to 4 days, 2 hours to 2 days, 2 hours to 24 hours, 6 hours to 24 hours, 10 hours to 24 hours, 15 hours to 20 hours, 20 hours to 40 hours, 6 hours to 40 hours, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0164] In some embodiments, the contacting or culturing is performed under static, flowing, stirring, shaking, agitating, or any combination thereof, such as 50-750 revolutions per minute (RPM), 50-650 RPM, 100-750 RPM, 100-700 RPM, 150-700 RPM, 200-750 RPM, 130-690 RPM, 90-720 RPM, 70-550 RPM, 110-710 RPM, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the contacting is performed under static conditions. In some embodiments, the contacting is performed under static conditions followed by contacting under flowing conditions. In some embodiments, the contacting is performed under flowing conditions followed by contacting under static conditions.

[0165] In some embodiments, the bacteria are contacted or cultured with the particles under shear stress conditions. In some embodiments, the solution, e.g., growth medium, imposes shear stress on the bacteria during the contacting or culturing step. In some embodiments, the term "shear stress" refers to the movement of the solution on the bacteria, e.g., on the bacteria attached to the particles. In some embodiments, the shear stress conditions include contacting or culturing the bacteria under conditions that include mixing, rocking, flowing, rotating, vortexing, stirring, shaking, agitating, foaming, pumping, bubbling, or any combination thereof.

[0166] In some embodiments, the terms "flow", "stirring", "shaking" and "agitating" refer to conditions that result in movement or transfer of the liquid phase within the culture vessel. In some embodiments, the movement is axial, radial, mixing, dispersing, or any combination thereof. The movement or transfer can be performed manually, automatically, or any combination thereof, by using mechanical means such as impellers, moving stages, rockers, shakers, etc. In some embodiments, the term "static conditions" refers to conditions in which no stirring or any other transfer action is performed on the liquid phase (manually, automatically, and / or mechanically).

[0167] In some embodiments, the contacting or culturing step comprises culturing under anaerobic conditions. In some embodiments, the term "anaerobic conditions" refers to conditions where the free oxygen is less than 500 ppm, 450 ppm, 400 ppm, 350 ppm, 300 ppm, 250 ppm, 200 ppm, 150 ppm, 100 ppm, 50 ppm, or 10 ppm, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, anaerobic conditions include conditions that do not provide free oxygen. In some embodiments, anaerobic conditions include conditions that lack free oxygen.

[0168] In some embodiments, the contacting or culturing step comprises culturing under aerobic conditions. In some embodiments, the term "aerobic conditions" refers to conditions that include the presence of molecular oxygen. In some embodiments, the oxygen concentration is greater than 20% (v / v relative to the total gas present in the culture).

[0169] In some embodiments, the growth medium further comprises an element that enhances adhesion of the bacteria to the particles.

[0170] In some embodiments, following attachment of the bacteria to the particles, some or all of the bacteria may detach and / or other bacteria in the suspension may adhere to the particles, to the particle-attached / bacteria, and / or to the matrix formed by the attached / adherent bacteria.

[0171] In some embodiments, the method includes a step of separating adherent and non-adherent bacterial fractions, e.g., to produce a composition comprising adherent and non-adherent bacteria. In some embodiments, the method further includes a step of removing bacteria that are not attached to particles from the solution, e.g., culture medium or growth medium. In some embodiments, the separating or removing step is performed at least one time point selected from the group consisting of before, during, or after the incubating, contacting, or culturing step, and any combination thereof. In some embodiments, the non-adherent / non-adherent bacteria are or include planktonic bacteria. In some embodiments, the non-adherent bacteria are devoid of sessile bacteria.

[0172] In some embodiments, separation of fractions or removal of non-adherent bacteria is performed by or includes filtration and / or gravity settling, e.g., centrifugation. In some embodiments, the term "filtration" includes any separation technique and any other process that utilizes a filter capable of separating fractions.

[0173] In some embodiments, the composition comprises particle-associated bacteria and / or bacteria in planktonic form. In some embodiments, the composition comprises bacteria attached to particles. In some embodiments, the composition comprises bacteria in planktonic form.

[0174] In some embodiments, the method includes removing metal carbonate from the particles, the particle-associated bacterial fraction, and / or from the solution in which the contacting or culturing is taking place, thereby creating a composition that includes trace amounts of metal carbonate.

[0175] In some embodiments, the step of removing the metal carbonate is performed at least one time during the preparation method, hi some embodiments, the removing step is performed at a time selected from (i) during contacting, e.g., during culturing, (ii) at the end of the contacting step, e.g., at the end of culturing, (iii) before separating the particle-attached bacteria from the non-attached bacterial fraction, (iv) after separating the particle-attached bacteria from the non-attached bacterial fraction, or (v) any combination of (i)-(iv).

[0176] In some embodiments, the removal step is performed in the same vessel in which the contacting is performed or in a different vessel. In some embodiments, the removal is performed on the particle-bound bacteria. In some embodiments, the removal step is performed on a precipitate fraction comprising the particle-bound bacteria. In some such embodiments, the precipitate can be resuspended, for example in saline, prior to removal.

[0177] In some embodiments, the term "removing metal carbonates" includes removing metal carbonates by removing acid and / or CO2 that is added to the solution during the contacting step, e.g., during the culturing step. 2The method includes partial or complete decomposition of the metal carbonate, depending on the amount of metal carbonate, etc. In some embodiments, 1% to 100% (by weight or by molar) of the initial calcium carbonate particles are removed. In some embodiments, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or even 100%, or any value and range therebetween, of the initial calcium carbonate particles are removed.

[0178] In some embodiments where the particles comprise a metal phosphate, e.g., DCP, the acid and / or CO 2 Addition of may result in partial or complete removal of DCP from the particles, the bacterial attached fraction and / or the solution in which contact or incubation takes place.

[0179] In some embodiments, the removing comprises adding an acid to the solution. 2 ), metal salts and water are produced. For example, (i) calcium carbonate (CaCO 3 ) is a metal carbonate compound, and (ii) the acid is HCl→carbon dioxide, calcium chloride (CaCl 2 ) and water are produced. In some embodiments, carbon dioxide gas is passed through the solution and released from the vessel in which the contacting takes place. (i) Magnesium carbonate (MgCO 3 ) is a metal carbonate compound and (ii) the acid is HCl→carbon dioxide. 2 ) and water are produced. In some embodiments, carbon dioxide gas is passed through the solution and released from the vessel in which the contacting takes place.

[0180] In some embodiments, the term "metal salt" refers to a compound composed of at least one anion and at least one cation. In some embodiments, the salt formed is solid at room temperature and water soluble at the temperature at which the contacting occurs.

[0181] In some embodiments, after removal of the metal carbonate, free metal ions are removed from the solution by adsorption, absorption, chelation, or any combination thereof.

[0182] In some embodiments, the term "acid" as used herein refers to an acid in the sense of the definition by Bronsted and Lowry. In some embodiments, the acid comprises acetic acid. In some embodiments, the acid is a halogen acid, such as hydrogen fluoride (HF), hydrogen chloride (HCl), hydrogen bromide (HBr), hydrogen iodide (HI), hydrogen astatine (HAt), hydrogen tennessine (HTs), or any combination thereof. In some embodiments, the acid comprises HCl, HBr, and HI.

[0183] In some embodiments, the removal is achieved by adding CO 2 This involves creating an environment enriched with calcium carbonate (CaCO 3 In those embodiments where calcium carbonate is a metal carbonate compound, calcium carbonate reacts with carbon dioxide and water to form calcium bicarbonate [(Ca(HCO 3 ) 2 ] is generated.

[0184] In some embodiments, the removing comprises contacting the composition with an effective amount of a chelator or chelating agent.

[0185] In some embodiments, the composition, particles and / or solution in which the contacting occurs contains trace amounts of metal carbonates after the removal / decomposition step. In some embodiments, during and / or after the removal step, an increase in the level of any of the decomposition products can be detected by any method known to one of skill in the art, such as by measuring salt concentrations, carbon dioxide and / or calcium bicarbonate levels in the container or solution in which the contacting occurs.

[0186] In some embodiments, the composition includes a trace amount of metal carbonate. In some embodiments, the term "trace amount" refers to a concentration ranging from 0.01 percent to less than about 5 percent. In some embodiments, the term "trace amount" refers to a metal carbonate concentration of less than about 5 percent of the total weight of the composition, such as less than 4 percent, less than 3 percent, less than 2 percent, less than 1 percent, or any value and range therebetween. In some embodiments, the term "trace amount" refers to a metal carbonate concentration of less than about 1 percent of the total weight of the composition.

[0187] In some embodiments, for example, acid and / or CO 2 The partial or complete dissolution of metal carbonate particles by HCl is dependent on the amount of acid and / or CO added to the solution relative to the moles of metal ions present in the particles, as will be appreciated by those skilled in the art. 2 In some embodiments, complete dissolution of the metal carbonate particles can be achieved by adding an acid to the solution in a molar ratio equal to the number of moles of metal ions present in the particles. (i) Calcium carbonate (CaCO 3In embodiments where (ii) the acid is HCl, and (iii) complete dissolution is desired, the mole to mole ratio between the HCl and calcium carbonate can be 2:1. In some embodiments, the mole to mole ratio between the HCl and calcium carbonate can be even lower, e.g., 1.5:1 or 1.25:1 or less. In some embodiments, the acid is added in an amount that does not affect the viability of the bacteria, e.g., the pH level during dissolution is suitable to allow for bacterial proliferation, growth and / or maintenance.

[0188] In some embodiments, the composition further comprises bacteria in aggregated form, for example after decomposition of metal carbonate particles. In some such embodiments, the majority of the adherent bacteria are attached to the surface of the polysaccharide-based material (e.g., more than 50% of the total adherent bacteria fraction, such as at least 51%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or even 100%, or any value and range therebetween). The total adherent bacteria can be determined by separating the two fractions as disclosed above.

[0189] In some embodiments, the term "bacteria in aggregate form" is interchangeable with the term "bacterial aggregates" and refers to a collection of individual bacterial particles held together. In some embodiments, the compositions of the present invention have an increased content of aggregated bacteria, e.g., at least 1.1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 9-fold, at least 10-fold, or more, or any value and range therebetween, compared to compositions prepared under the same contact / culture conditions and in the absence of particles and / or in the presence of particles currently used for bacterial growth.

[0190] In some embodiments, the composition comprises bacteria in the form of a biofilm. In some embodiments, the biofilm is in the form of a dry biofilm or in a solid form, such as a powder. In some embodiments, the term "biofilm" refers to a community of bacteria embedded in a matrix that includes self-produced exopolysaccharides, for example attached to the surface of a particle. In some embodiments, planktonic bacteria are attached, trapped, incorporated, or embedded under, on, or within the biofilm.

[0191] In some embodiments, the bacterial load of the composition, or at the end of the contacting or culturing step, is at least 1 E per gram, e.g., per gram of particles provided, added, or used in the methods of the invention, per total weight of bacteria at the end of the culturing, and / or per total weight of the composition. 4 pieces, e.g. at least 1·E 6 In some embodiments, the method further comprises harvesting the cultured bacteria. In some embodiments, the harvested cultured bacteria has at least 1·E per gram, e.g., per gram of particles, and / or per total weight of the harvested bacteria. 4 pieces, e.g. at least 1·E 6 The bacterial load is

[0192] In some embodiments, the bacterial load is at least 1 E 4 ~At least 1 E 20 pcs, at least 1·E 5 ~At least 1 E 15 pcs, at least 1·E 5 ~At least 1 E 10 pcs, or at least 1·E 7 ~At least 1 E 9 In some embodiments, the bacterial load spans a range of at least 1 E 6 pcs, at least 1·E 7 pcs, at least 1·E 8 pcs, at least 1·E 9 pcs, at least 1·E10 pcs, at least 1·E 11 pcs, at least 1·E 12 pcs, at least 1·E 14 pcs, at least 1·E 16 pcs, or at least 1·E 20 or any value and range therebetween. Each possibility represents an independent embodiment of the present invention. In some such embodiments, the amount of bacteria can be per gram, for example, per gram of particles provided, added, or used in the method, per total weight of cultured bacteria at the end, and / or per gram of composition.

[0193] In some embodiments, the determination of bacterial load is performed by any standard method known and available to those of skill in the art, non-limiting examples of which include, but are not limited to, plate counting, spectrophotometric (turbidity) analysis, and the like.

[0194] In some embodiments, the bacterial load is determined by viable colony forming units (CFU), quantitative polymerase chain reaction (qPCR), flow cytometry, live-dead staining, propidium monoazide qPCR (PMA-qPCR), microscopy, metabolic assays, other common methods, or any combination thereof.

[0195] In some embodiments, the method further comprises contacting or culturing at least one additional microorganism. In some embodiments, the composition further comprises at least one additional microorganism. In some embodiments, the at least one additional microorganism is selected from fungi, bacteriophages, viruses, archaea, etc., or any combination thereof (e.g., originating or derived from the biological sample, found in, constituting, or known to reside in the environmental niche of interest).

[0196] In some embodiments, the particles are dispersed in the composition or in the carrier that contains the composition. In some embodiments, the term "dispersed" in reference to the presence of particles in the composition or carrier includes that the particles are present throughout substantially the entire composition or carrier, without being substantially concentrated in any part of the composition or carrier. In addition, the term "dispersed" also includes that the particles are present in a localized area of ​​the composition or carrier.

[0197] In some embodiments, the compositions and / or methods of the invention include a population of a single bacterial strain or a mixture of two or more strains. In some such embodiments, the different strains are contacted or cultured with the particles separately or together as a co-culture, or both are mixed. The bacterial strains and / or the ratio between the different strains can vary, for example, depending on the intended application. In some embodiments, the strains and ratios can be selected according to the desired characteristics of the strains, for example, the ability to reduce the pH level in the target area, the ability to inhibit the growth of a particular pathogenic bacteria, etc. In some embodiments, the different strains are isolated / originate from different sources, for example, different donors. In some embodiments, when the bacteria originate from a sample, for example, a donor, the compositions and / or culture methods can be enriched or supplemented with additional bacteria.

[0198] According to some embodiments, compositions produced according to the methods of the invention are provided.

[0199] According to some embodiments, there is provided a pharmaceutical composition comprising a composition disclosed herein and an acceptable carrier or excipient.

[0200] In some embodiments, the term "pharmaceutical composition" encompasses the terms "dietetic composition" and "nutritional composition."

[0201] In some embodiments, the terms "dietary composition", "nutritional composition", and "nutraceutical composition" refer to a composition suitable for consumption as a dietary supplement, e.g., to supplement a normal diet, correct a nutritional deficiency, maintain an adequate intake of certain nutrients, and / or support a particular physiological function.

[0202] In some embodiments, the composition of the present invention is administered to a subject immediately after culturing. In some embodiments, the composition is administered after storage, for example, at room temperature, a temperature in the range of 2-8°C, or a temperature below -18°C. In some embodiments, the composition is provided in a solid form, for example, lyophilized, spray-dried, or frozen. In some embodiments, the solid compositions disclosed herein are stable for at least 3 months at room temperature (e.g., a temperature selected from the group consisting of about 20, 21, 22, 23, 24, and 25°C). In this context, the term "solid" refers to the physical state of a material. In some embodiments, the composition is a solid composition. In some embodiments, the composition is a lyophilized composition.

[0203] Typically, the terms "lyophilization" and "freeze-drying" are interchangeable and refer to freezing a solution and then reducing the concentration of water, for example by sublimation, to a level that does not support biological and / or chemical reactions. The resulting lyophilized composition can be stored for an extended period of time while maintaining its stability. In some embodiments, the lyophilized composition can be used as a solid. In some embodiments, the solid or composition can be placed in a suitable delivery vehicle, such as a fat-based carrier, for use as a suppository, or can be reconstituted by adding a semi-liquid or liquid solution. The volume added during reconstitution can be the same, less, or more compared to the initial volume of the solution before the lyophilization process.

[0204] In some embodiments, the composition is a composition for pharmaceutical use. In some embodiments, the composition is a composition for agricultural use. In some embodiments, the composition is a composition for veterinary use. In some embodiments, the composition is a composition for use as a non-prescription drug, for example as a supplement.

[0205] In some embodiments, the pharmaceutical composition is for use in treating or preventing dysbiosis in a subject in need thereof. In some embodiments, the compositions disclosed herein are for use in the manufacture of a medicament for treating or preventing a dysbiosis-associated condition.

[0206] In some embodiments, the composition is a synthetic composition. In some embodiments, the term "synthetic composition" refers to a composition that includes bacteria grown or cultured in vitro. In some embodiments, a synthetic composition includes an artificial composition. In some embodiments, a synthetic composition is a man-made composition, such as, but not limited to, a composition created or produced in a laboratory and / or manufacturing site or facility. In some embodiments, a synthetic composition does not include a composition that is isolated or obtained from nature itself. In some embodiments, the composition is frozen, spray-dried, or freeze-dried. In some embodiments, the composition is in the form of a dry powder. In some embodiments, the composition includes a cryoprotectant, a lyoprotectant, an antioxidant, or any combination thereof. In some embodiments, the composition includes at least one metabolic product produced in vitro by at least one bacterium.

[0207] In some embodiments, a method is provided for preventing or treating a dysbiosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition of the invention.

[0208] The term "therapeutically effective amount" or "effective amount" refers to the amount necessary to prevent, ameliorate and / or treat a disease, disorder or condition. The effective dose may vary depending on the subject's sex, age and weight, the disease or condition and its severity, and any other factors that may be recognized by a person skilled in the art.

[0209] The compositions and formulations disclosed herein can be used internally or externally, e.g., to treat or prevent dysbiosis. In some embodiments, the compositions and formulations can be administered to the surface of a body part of a subject in need thereof.

[0210] As used herein, the term "surface of a body part of a subject" refers to external surfaces of the body that are visible to the naked eye, such as the skin of the face, throat, scalp, chest, back, ears, neck, hands, elbows, knees, and other skin sites, as well as surfaces of internal parts that are part of the internal structure of an individual, such as, but not limited to, the oral cavity, digestive tract, and lower reproductive tract, such as, but not limited to, the vagina.

[0211] As used herein, the term "dysbiosis" is characterized by an alteration, imbalance, damage and / or dysfunction of the microbiota in any tissue and / or surface of any body part of an organism, including but not limited to any external or internal surface of the body and the deeper layers of the skin, that is typically associated with a disease, health condition or clinical condition. The imbalance can be an imbalance of any microbial community, such as, but not limited to, the digestive tract, skin, mouth, bronchi, vagina or rectum.

[0212] In some embodiments, the dysbiosis is a tumor dysbiosis. In some embodiments, the composition is used to treat gastrointestinal or intestinal dysbiosis. In some embodiments, the composition is used to treat skin and tissue dysbiosis. In some embodiments, the composition is used to treat dysbiosis associated with interstitial cystitis, polycystic ovary syndrome. In some embodiments, the dysbiosis is vaginal and female genital dysbiosis, such as bacterial vaginosis, urinary tract infection, human papilloma virus infection, urogenital infection, urinary tract infection, candida infection, infertility, sexually transmitted diseases, and gynecological cancer. In some embodiments, the composition is used to treat male genital dysbiosis. In some embodiments, the composition is used to reduce and / or prevent dysbiosis associated with premature birth and miscarriage.

[0213] In some embodiments, the dysbiosis comprises an imbalance in the microbiota. In some embodiments, the pharmaceutical composition is a composition for use in modulating the microbiota in a subject in need of such modulation. In some embodiments, the compositions disclosed herein are compositions for use in the manufacture of a medicament for modulating the microbiota. In some embodiments, a method is provided for modulating the microbiota in a subject in need of such modulation, comprising administering a therapeutically effective amount of a composition of the present invention to the subject. In some embodiments, the microbiota comprises vaginal flora, gut flora, or skin flora.

[0214] In some embodiments, the term "modulating the microbiota" includes suppressing or reducing the abundance of bacteria typically associated with a disease, health condition or clinical condition, restoring a healthy microbiome balance, increasing the diversity and / or achieving colonization of beneficial bacteria typically associated with a disease, health condition or clinical condition in any tissue and / or surface of any body part of an organism, including, but not limited to, any external or internal surface of the body and the deeper layers of the skin.

[0215] In some embodiments, the term "treating a dysbiosis" includes generating a desired / predetermined microbial profile in a subject's environmental niche that is beneficial for a purpose, such as increasing the subject's responsiveness to an administered drug.

[0216] In some embodiments, a "microbial profile" comprises bacterial diversity (e.g., α-diversity or β-diversity), relative bacterial abundance, and / or bacterial load.

[0217] In some embodiments, the terms "bacterial load," "bacterial viability," and "bacterial count" are interchangeable. In some embodiments, the composition comprises live bacteria and the bacterial count is measured by CFU.

[0218] In some embodiments, the amount of bacteria in the composition produced according to the present invention is calculated per gram of particles in dry form introduced into the culture medium or culture vessel. In some embodiments, the amount of bacteria is calculated per gram of particles in dry form used to contact with bacteria. In some embodiments, when measuring the amount of bacteria in the composition, the weight of the particles added to the culture system is taken into account. In some embodiments, when measuring the amount of bacteria in the composition, the weight of the particles in the final composition is taken into account.

[0219] In some embodiments, the altered microbiota comprises a pathogenic microorganism. In some embodiments, the altered microbiota is associated with a medical condition and is detrimental to the health of the subject, e.g., to a human subject.

[0220] As used herein, the term "altered microbiota" refers to a microbiota that deviates from the homeostatic microbiota, for example in an unhealthy subject.

[0221] In some embodiments, the subject is a mammal. In some embodiments, the subject is an animal. In some embodiments, the subject is a human subject. The subject may be male or female.

[0222] In some embodiments, a subject in need of the treatment described herein is suffering from or at risk of suffering from a disease, disorder or condition.

[0223] In some embodiments, the composition includes a carrier or excipient. In some embodiments, the carrier is a veterinary, agricultural and / or pharmaceutically acceptable carrier. In some embodiments, the term "carrier", "excipient" or "adjuvant" refers to any component of a pharmaceutical composition that is not an active agent. As used herein, the term "pharmaceutically acceptable" carrier, solvent, diluent, excipient and vehicle generally refers to a non-toxic, inert solid, semi-solid, liquid filler, diluent, encapsulating material, any type of formulation auxiliary, or simply a sterile aqueous solution, e.g., saline. In some embodiments, the term "pharmaceutically acceptable carrier" refers to any diluent or vehicle suitable for use in humans or other animals. Some examples of materials which can function as pharma- ceutically acceptable carriers are sugars such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate, powdered tragacanth, malt, gelatin, talc, excipients such as cocoa butter and suppository waxes and hard fats, oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil, glycols such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol, esters such as ethyl oleate and ethyl laurate, agar, buffers such as magnesium hydroxide and aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol and phosphate buffers, and other non-toxic compatible substances used in pharmaceutical formulations.Some non-limiting examples of materials that can function as carriers herein include sugars, starches, cellulose and its derivatives, powered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer, cocoa butter (suppository base), emulsifiers (e.g., carbomer, hydroxypropylcellulose, sodium lauryl sulfate), and other non-toxic pharmaceutically compatible materials used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as colorants, flavorings, excipients, stabilizers, antioxidants, and preservatives, may also be present. Any non-toxic, inert, and effective carrier can be used to formulate the compositions contemplated herein. In this respect, suitable pharma- ceutically acceptable carriers, excipients and diluents are well known to those skilled in the art, such as those described in "The Merck Index," 13th Edition, edited by Budavari et al., Merck & Co., Inc., Rahway, NJ (2001), CTFA (Cosmetic, Toiletry, and Fragrance Association) "International Cosmetic Ingredient Dictionary and Handbook," 10th Edition (2004), and "Inactive Ingredient Guide," U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the contents of all of which are incorporated herein by reference in their entirety.Examples of pharma-ceutically acceptable carriers, excipients and diluents useful in the compositions of the present invention include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution and DMSO.These additional inactive ingredients and effective formulation and administration procedures are well known in the art and are described in standard textbooks such as "Goodman and Gillman's: The Pharmacological Bases of Therapeutics," 8th Edition, edited by Gilman et al., Pergamon Press (1990), "Remington's Pharmaceutical Sciences," 18th Edition, Mack Publishing Co., Easton, PA (1990), and "Remington: The Science and Practice of Pharmacy," 21st Edition, Lippincott Williams & Wilkins, Philadelphia, PA (2005), each of which is incorporated herein by reference in its entirety. The compositions described herein may be included in artificially created structures such as liposomes, ISCOMs, sustained release particles, and other vehicles. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. Liposomes for use with the peptides described herein are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and sterols, such as cholesterol. A variety of methods are available for preparing liposomes, as reviewed, for example, in Coligan, JE et al., Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York. See also U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369. The carrier may comprise from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0224] Pharmaceutical compositions may take any physical form necessary for proper administration. The compositions may be administered in any suitable form, including, but not limited to, liquid form, gel form, semi-liquid form (e.g., liquids, such as viscous liquids, with some solids), semi-solid form (solids with some liquids), or solid form. The compositions may be provided, for example, in the form of tablets, pessaries, creams, suppositories, capsules, liquids, foods, chewables, non-chewables, transmucosal oral preparations, sublinguals, sustained release, non-sustained release, sustained release, or non-sustained release forms.

[0225] In some embodiments, the composition is formulated for administration by a mode selected from the group consisting of rectal administration, parenteral administration, mucosal administration, vaginal administration, nasal administration, topical administration, external use, pulmonary administration, ocular administration, oral administration, oral mucosal administration, and any combination thereof.In some embodiments, the composition is administered orally.In some embodiments, the composition is administered topically (e.g., by inserting a suppository, capsule or other suitable form into the vagina).

[0226] In some embodiments, the compositions may be placed into various drug delivery systems, including, but not limited to, capsules, suppositories, dissolvable shells, and the like.

[0227] In some embodiments, the composition is provided in the form of a liquid or semisolid, cream, suppository, pessary, tablet, pill, caplet, capsule, granule, ointment, lotion, gel, spray, sachet, drops, oral solution, suspension, syrup, emulation, oil or film.

[0228] In some embodiments, the composition comprises a carrier and at least one cultured bacterial species disposed within the carrier. In some embodiments, the composition comprises a mixture of dry bacteria. In some embodiments, each bacterium is cultured separately. In some embodiments, the composition comprises co-cultured bacteria.

[0229] In some embodiments, the composition is disposed within an enteric coating.

[0230] In some embodiments, the composition is configured for pH-dependent targeted release of the bacterial strain in the digestive tract. In some embodiments, the composition is coated with a compound, such as alginate, and configured to release the bacteria at a pH found in the gut of a subject.

[0231] General rules In this specification, all numerical values ​​are assumed to be modified by the term "about." The term "about" generally refers to a range of numbers that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In many cases, when the term "about" is in front of a numerical value, it is intended to indicate ±10%.

[0232] The words "comprises," "comprising," "includes," "including," "having" and their conjugations mean "including, but not limited to."

[0233] The term "consisting of" means "including and limited to."

[0234] The term "consisting essentially of" means that a composition, method, or structure may include additional ingredients, steps, and / or components, but only if those additional ingredients, steps, and / or components do not materially alter the basic and novel characteristics of the composition, method, or structure of the present application.

[0235] The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any embodiment described as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments and / or to exclude the incorporation of features from other embodiments.

[0236] The term "optionally" is used herein to mean "is provided in some embodiments and is not provided in other embodiments." Any particular embodiment of the invention may include multiple "optional" features, unless those features conflict.

[0237] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As used in this specification and the appended claims, the term "or" is generally used in its sense including "and / or" unless the content clearly dictates otherwise.

[0238] Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0239] Whenever a numerical range is given herein, it is meant to include any stated number (fractional or integer) within the stated range. The phrases "range between" a first stated number and a second stated number and "range from" a first stated number to a second stated number are used interchangeably herein and are meant to include the first and second stated numbers and all fractional and integer numbers therebetween. For example, a statement "range between 15% and 85%" should be considered to include the first and second stated percentages and all numbers within the stated range. The phrases "at least," "up to," and "less than" used herein are meant to include the stated numbers. For example, "up to 85%" should be considered to include the stated numbers.

[0240] As used herein, the term "method" refers to methods, means, techniques and procedures for accomplishing a given task, including, but not limited to, methods, means, techniques and procedures that are known to or are readily developed from known methods, means, techniques and procedures by those of skill in the art of chemistry, pharmacology, biology, biochemistry, microbiology and medicine.

[0241] It will be understood that certain features of the invention that are described in the context of separate embodiments for clarity of description may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described in the context of a single embodiment for clarity of description may also be provided separately or in any suitable subcombination, or in any other described embodiment of the invention, as appropriate. Certain features described in the context of various embodiments should not be considered essential features of those embodiments, unless the embodiment is inoperable without those elements.

[0242] The description of various embodiments of the present invention is presented for illustrative purposes, but is not intended to be exhaustive or limited to the disclosed embodiments. Many changes and modifications that do not depart from the scope and spirit of the described embodiments will be apparent to those skilled in the art. The terms used in this specification are selected to best explain the principles of the embodiments, practical applications or technical improvements to the technology found in the market, or to enable those skilled in the art to understand the embodiments disclosed in this specification.

[0243] Various embodiments and aspects of the present invention as delineated above and as claimed in the claims section below are found experimentally supported in the following examples.

[0244] Other terms used herein are defined as known in the art.

[0245] Additional objects, advantages, and novel features of the present invention will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated above and as claimed in the claims section below finds experimental support in the following examples. EXAMPLES

[0246] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques, which are fully explained in the literature.For example, "Molecular Cloning: A laboratory Manual" by Sambrook et al. (1989); "Current Protocols in Molecular Biology" Vols. I-III, edited by Ausubel, RM (1994); "Current Protocols in Molecular Biology" by Ausubel et al., John Wiley and Sons, Baltimore, Maryland (1989); "A Practical Guide to Molecular Cloning" by Perbal, John Wiley & Sons, New York (1988); "Recombinant DNA" by Watson et al., Scientific American Books, New York; and "Genome Analysis: A Laboratory Manual Series" Vols. 1-4, edited by Birren et al., Cold Spring Harbor Laboratory. Press, New York (1998); the methods described in U.S. Pat. Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659 and 5,272,057; Cell Biology: A Laboratory Handbook, Vols. I-III, Cellis, JE ed. (1994); Culture of Animal Cells-A Manual of Basic Technique, Freshney, Wiley-Liss, NY (1994), 3rd Edition; Current Protocols in Immunology, Vols. I-III, Coligan JE ed. (1994); Basic and Clinical Immunology, Stites et al. (eds.), 8th Edition, Appleton & Lange, Norwalk, Connecticut (1994); Selected Methods in Cellular Immunology, W.H. Freeman and Co., New York (1980). Available immunoassays are described extensively in the patent and scientific literature.See, for example, U.S. Patent Nos. 3,791,932, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517, 3,879,262, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, 4,098,876, 4,879,219, 5,011,771 and 5,281,521, "Oligonucleotide See, for example, "Nucleic Acid Hybridization," edited by Gait, MJ (1984); "Nucleic Acid Hybridization," edited by Hames, BD and Higgins SJ (1985); "Transciption and Translation," edited by Hames, BD and Higgins SJ (1984); "Animal Cell Culture," edited by Freshney, RI (1986); "Immobilized Cells and Enzymes," IRL Press, (1986); "A Practical Guide to Molecular Cloning," Perbal, B. (1984); and "Methods in Enzymology," vols. 1-317, Academic Press, "PCR Protocols: A Guide To Methods And Applications," Academic Press, San Diego, Calif. (1990); Marshak et al., "Strategies for Protein Purification and Characterization--A Laboratory Course Manual," CSHL Press (1996). All of these publications are incorporated herein by reference. Other general references are provided throughout this document.

[0247] In the following examples, in order to illustrate the advantages of the method of the present invention for producing a composition comprising a population of at least one bacterial strain at least partially attached to the particles, when using the particles as described herein, as compared to other cultivation methods, various bacterial strains having different characteristics, e.g., different taxa and different origins, were cultivated as detailed in Table 1 below as embodiments. In the examples, MCC and CaCO are used as embodiments of polysaccharide-based materials and metal carbonate particles. 3 was used. material and method Bacteria tested [Table 1]

[0248] Particles used Microcrystalline cellulose (MCC) and CaCO 3 (CAS number: 1-34-471) was used. All particle weight and ratio values ​​in the Examples section refer to particles in dry, non-hydrated form. The moisture content within the particles was less than 5%. The particles can range in diameter from 0.1 microns to 500 microns.

[0249] Culture conditions and preparation of bacterial compositions The cultures were incubated with 40 g of (i) MCC particles or (ii) various ratios (w / w) of MCC and CaCO 3 The experiments were performed in sterile bottles containing 200 ml of growth medium with or without particles, as detailed in the "Experimental Procedures" section below.

[0250] Tested MCC and CaCO 3 The ratios are 75%:25% (30g:10g), 50%:50% (20g:20g), and 25%:75% (10g:30g).

[0251] 100% CaCO 3 Alternatively, 100% MCC particles were also tested.

[0252] Or MCC / CaCO 3Particle mixtures containing / DCP may also be used.

[0253] The media used for the cultures contained yeast extract, peptone, Tween, L-cysteine, sodium, potassium, manganese and magnesium salts, and +(D)-glucose for Lactobacillus crispatus and Lactobacillus paracasei, and Bifidobacterium broth supplemented with L-cysteine, Tween and +(D)-glucose for Bifidobacterium bifidum.

[0254] Planktonic growth (cultivation in the absence of particles) was also performed under corresponding conditions as detailed for each bacterium.

[0255] If the cultivation was carried out in the presence of particles, the particles were premixed with the medium (pH of the medium was adjusted according to the bacteria used - see conditions below) before mixing with the inoculum.

[0256] Experimental procedure Cultures of each bacteria were prepared from an initial inoculum stored at -80°C (stock inoculum contained planktonic bacteria with a 50% glucose solution in a 5:1 w / v ratio).

[0257] Add 50 μl (for Lactobacillus clipatus and Lactobacillus paracasei) or 100 μl (for Bifidobacterium bifidum) of inoculum to 40 g of pelleted (100% MCC, 100% CaCO 3 , or MCC / CaCO as specified above 3 The cultures were mixed with 200 ml of medium with or without the addition of the 100 ml medium (Lactobacillus crispatus and Lactobacillus paracasei) or 20 hours (Bifidobacterium bifidum) under the following conditions: pH range of approximately 5.0–6.5, under anaerobic conditions (gas mixture: N 2 -90%, H 2 -5%, CO 2 -5%), temperature 37°C, and stirring with a magnet at approximately 25 RPM for the entire incubation period.

[0258] In the examples, CaCO was added to the solution of acid. 3 The particles were removed from the bacterial composition.

[0259] To quantitatively evaluate bacterial adhesion to the different particles tested, an assessment of the total bacterial count [expressed in Log(CFU / ml)] was performed as follows: - at the end of the culture for (i) planktonic growth, i.e., culture without particles (Figure 1, "Planktonic"), and (ii) culture in the presence of particles (total bacterial mass, i.e., particle-attached and non-attached bacterial fractions combined; in Figure 1, labeled "Group 1"). Planktonic cultures were used as the reference group; and - in the isolated particle-bound bacterial fraction (labeled "Group 2" in Figure 1). The isolation procedures for each group tested were performed as detailed below. By comparing the bacterial counts in the different adherent fractions (shown in Group 2), CaCO 3 The change in adhesion levels when using particle mixtures according to the invention versus culturing in the presence of MCC without particles is shown.

[0260] To evaluate the feasibility of particle dissolution using acid, CaCO from the particle-attached bacterial fraction was 3 After particle removal / titration, bacterial counts (CFU) were assessed (labeled “Group 3” in Figure 1 ).

[0261] The results in Figure 1 show the bacterial counts (Log CFU / ml) obtained for groups 1–3 and planktonic cultures for Bifidobacterium bifidum.

[0262] All methods were performed as specified below.

[0263] Measurement of bacterial load (colony forming units, CFU) Unless otherwise indicated, CFU determinations were performed as follows: samples were taken from each culture bottle and transferred to three sterile tubes (three for each culture condition, 10 ml each).

[0264] For group 1, vortexing was performed at high speed for 2 min to dislodge the bacteria bound to the particles. To assess adhesion, CFU determinations (drop assay) were performed from each condition tested by serial dilution and bacteria were plated in triplicate on agar plates. Plates were placed under anaerobic conditions (gas mixture: N 2 -90%, H 2 -5%, CO 2 -5%) at 37°C for 24 to 48 hours, after which CFU counts were performed.

[0265] For group 2, the particle-attached bacterial fraction was separated from the non-attached fraction by centrifugation (48×g for 3 min at 21±2° C.), removal of the supernatant, and washing the pellet containing the particle-attached bacterial fraction with 0.9% saline. Centrifugation was repeated under the same conditions, and the supernatant was removed to obtain a pellet of the particle-attached bacterial fraction (approximately 5 g). The pellet was resuspended in 5 ml of 0.9% saline, and the drop assay was performed as detailed above for group 1.

[0266] For group 3, the particle-associated bacterial fraction was separated from the non-associated fraction by centrifugation (3 min at 48 × g at 21 ± 2 °C), removing the supernatant, and washing the pellet containing the particle-associated bacterial fraction with 0.9% saline. Centrifugation was repeated under the same conditions, and the supernatant was removed to obtain a pellet of particle-associated bacterial fraction (approximately 5 g). The pellet was resuspended in 5 ml of 0.9% saline and diluted with CaCO3 with HCl as detailed below. 3 The particles were removed and the drop assay was performed as detailed for group 1.

[0267] For planktonic growth, aggregates were detached / dissociated by vortexing at high speed for 2 min and the drop assay was performed.

[0268] CaCO using acid 3 Particle Dissolution / Removal For Lactobacillus clipatus, Lactobacillus paracasei and Bifidobacterium bifidum, 100–200 μl of 5 M HCl was gradually added to the saline (0.9%) resuspended fractions of Group 3 prepared as detailed above while the samples were being manually mixed. The exact volumes added for each culture condition are shown in Table 2.

[0269] In general, HCl was added taking into account two criteria: 1- CaCO in progress 3 CO from solution, indicating particle dissolution reaction 2 Emissions, and 2-CaCO 3 pH level throughout the lysis process to ensure bacterial viability and functionality at the end of particle lysis.

[0270] Several HCl volumes were tested. The volumes listed in Table 2 are those that provide the greatest CaCO concentration with minimal impact on bacterial counts. 3 Represents particle lysis (bacterial counts were determined for each volume added. Figure 1 shows the bacterial counts in the volumes shown in the table for Bifidobacterium bifidum). [Table 2]

[0271] H 2 O 2 Resistance of the attached bacterial fraction to stress H 2 O 2 The advantage of a composition comprising particle-attached bacteria prepared according to the present invention in withstanding stress conditions was investigated in comparison with a bacterial composition comprising suspended bacteria and / or attached bacteria (96w / p culture).

[0272] H 2 O 2 Several compositions were tested for bacterial resistance to: (i) a composition prepared according to an embodiment of the present invention [MCC:CaCO in a ratio of 1:1 (w / w)]; 3(ii) bacterial compositions prepared on 96-well plates (labeled "96w / p culture"), and (iii) bacterial compositions in suspension (labeled "suspended"). Compositions (i) and (iii) were tested in wet form (at the end of the culture) and after freeze-drying. Composition (iii) prepared in 96w / p was examined in wet form. To measure the stress after freeze-drying, the dried composition was reconstituted in PBS for 15 minutes at room temperature (about 25°C). Both the wet and freeze-dried forms were treated in the same manner as detailed below.

[0273] Resistance experiments were performed with Lactobacillus plantarum. Starting bacterial numbers were similar in all test conditions.

[0274] 96w / p culture procedure As a comparison for the advantage of compositions according to embodiments of the invention in withstanding stresses, a culture at 96w / p was used, where bacterial growth occurs in an attached form.

[0275] A 96w / p culture of Lactobacillus plantarum was prepared from a primary inoculum stored at -80°C [the stock inoculum contained planktonic bacteria in a 5% (v / v) DMSO solution].

[0276] First, 50 μl of the inoculum was mixed with 10 ml of Lactobacillus plantarum medium (yeast extract, Tween, L-cysteine, sodium, potassium, manganese and magnesium salts, and +(D)-glucose) and incubated under anaerobic conditions (gas mixture: N 2 -90%, H 2 -5%, CO 2 -5%) and incubated / cultured at 37°C for 10 hours.

[0277] After 10 hours of incubation, OD 600 Measure the bacterial population at an initial OD of 0.01 in Lactobacillus plantarum medium. 600 and incubated for an additional 12 hours under the same conditions with shaking at 100 RPM.

[0278] After 12 hours of incubation, OD 600 Measure the bacterial population at an initial OD of 0.01 in Lactobacillus plantarum medium. 600 Fresh bacterial stocks were transferred in triplicate to 96w / p (100 μl per well) and incubated under static conditions, under the same conditions detailed above, for approximately 24 hours.

[0279] After incubation, the spent solution was aspirated and the cultures were gently washed with PBS to remove the floating bacterial fraction (not attached to the 96w / p). The supernatant was aspirated from the well plate. In the next step, the H 2 O 2 The bacterial population attached to the plate was cultured by adding medium containing a series of increasing concentrations of H. 2 O 2 was exposed to.

[0280] Cultivation of bacteria according to embodiments of the present invention in a fermentor The cultivation was carried out in a 5 L fermenter containing 2 L of growth medium, with 400 g of a particle mixture [MCC and CaCO in a 1:1 (w / w) ratio]. 3 (200 g:200 g)] as detailed in "Fermentation Procedure" below. The particles were premixed with the medium (pH of the medium was adjusted to suit the culture conditions - see below) before the inoculum was added to the fermenter.

[0281] The medium used for the culture contained yeast extract, peptone, Tween, L-cysteine, sodium, potassium, manganese and magnesium salts, and +(D)-glucose.

[0282] Oxidative stress is a well-accepted assay and was chosen as an exemplary stress condition to examine the benefits of compositions comprising particle-attached bacteria prepared according to the present invention.

[0283] Fermentation Procedure Cultures of Lactobacillus plantarum were prepared from an initial inoculum stored at -80°C (the stock inoculum contained concentrated planktonic bacteria with a 50% glucose solution in a 5:1 w / v ratio).

[0284] 100 μl of inoculum was mixed with 300 ml of medium and incubated for 10 h under the following conditions: pH range of approximately 5.0–6.5, temperature of 37°C, under anaerobic conditions (gas mixture: N 2 -90%, H 2 -5%, CO 2 -5%), no stirring.

[0285] A fermenter containing 2 L of medium and 400 g of the particle mixture was inoculated with 50 ml of culture. The fermentation / cultivation was carried out for 15 hours with constant stirring. The initial pH was 6.9. The final pH was set to 5.94 and the temperature was set to 37°C.

[0286] Suspension growth was carried out under corresponding conditions but in the absence of particles.

[0287] In preparation for stress measurements of the compositions of the invention, after incubation, the particle-bound fraction was separated from the non-bound fraction (e.g., floating phase) by centrifugation (3 min at 48×g at 21±2° C.), the supernatant was removed, followed by a further PBS wash and centrifugation (5 min at 4,248×g at 21±2° C.). The supernatant was removed and the pellet was eluted in H 2 O 2 I was under stress.

[0288] Suspension cultures were prepared for stress measurements by centrifugation to remove spent medium, followed by a further wash with PBS and a further centrifugation (both at 4,248 × g for 5 min at 21 ± 2°C) before stress.

[0289] H 2 O 2 stress Bacteria were exposed to different concentrations of H 2 O 2Exposed to: 0.1, 0.2 or 0.5% for 1 min. 2 O 2 Concentrations were prepared in Lactobacillus plantarum culture medium.

[0290] H 2 O 2 To stop the induction stress, 0.2% sodium thiosulfate in PBS was added to the cultures.

[0291] To quantitatively assess bacterial survival after stress, bacteria grown in 96w / p were detached from the surface of the wells by vigorous pipetting. 3 Bacteria grown in the presence of were detached from the particles by vortexing (1.5 min at high speed). Planktonic cultures were also vortexed. Total bacterial counts, expressed as colony forming units (CFU), were determined using serial dilutions in PBS and plating on MRS plates. Results are expressed as a percentage compared to the control for each group (see "H 2 O 2 "Additive-free" was considered to be 100%.

[0292] Example 1 MCC and CaCO 3 Cultivation of bacterial populations in the presence of particles In the following examples, MCC and CaCO 3 To investigate the level of attachment during cultures using a particle mixture with MCC particles. For this purpose, cultures in the presence of the particle mixture were cultured with MCC particles alone or with CaCO. 3 The results were compared to cultures in the presence of particles only. In these experiments, suspension cultures without particles were carried out as a reference.

[0293] Three different bacteria, Bifidobacterium bifidum, Lactobacillus paracasei and Lactobacillus clipatus, were cultured separately on the particle mixtures with different ratios, and on pure MCC or pure CaCO. 3The particles were used to culture different bacteria as detailed above in the sections "Culture conditions and preparation of bacterial compositions" and "Experimental procedures". After cultivation, the adhesion levels were tested and compared between the different test groups.

[0294] Figure 1 shows the bacterial counts (CFU / ml) of Bifidobacterium bifidum grown without particles (planktonic growth morphology, labeled "planktonic"), the bacterial counts of the same bacteria grown in the presence of particles in the combined particle-associated and non-attached bacterial fractions (labeled "Group 1"), the bacterial counts in the particle-associated bacterial fraction alone (labeled "Group 2"), and the bacterial counts after partial removal of metal carbonate particles from the particle-associated bacterial fraction (i.e., from Group 2) by acid (labeled "Group 3").

[0295] The results show that the cultivation of Bifidobacterium bifidum in the presence of a particle mixture resulted in increased attachment compared to growth in the presence of 100% MCC particles (compare in Figure 1 Group 2, culturing in the presence of 100% MCC particles versus MCC and CaCO). 3 Cultivation in the presence of both results in an increase from Log 7.92 CFU / ml to Log 8.68-8.87 CFU / ml).

[0296] The results also show that the cultivation of Bifidobacterium bifidum in the presence of the particle mixture resulted in an increase in the total bacterial count - Log 9.35 to 9.49 CFU / ml (i.e., particle-attached and non-attached bacterial fractions, group 1) compared to other bacterial growth forms - planktonic cultures without particles (Log 8.10 CFU / ml) or cultivation in the presence of 100% MCC particles (Log 8.49 CFU / ml) (Figure 1).

[0297] Cultures of Lactobacillus crispatus and Lactobacillus paracasei also supported these results, demonstrating a trend towards increased total bacterial counts and levels of adhesion when cultured on particle mixtures (using the concentrations detailed above) compared to cultures on 100% MCC particles, combining particle-attached and non-attached bacterial fractions.

[0298] These results are consistent with those of 100% CaCO without MCC particles. 3 It has also been demonstrated that the cultivation of various bacteria in the presence of particles resulted in a viscous culture medium, which generally required an increase in the stirring speed to obtain a homogenous culture medium, thus negatively affecting the level of bacterial adhesion to the particles.

[0299] CaCO 3 It can also be seen that the addition of acid to the composition containing the -MCC particle mixture did not significantly affect the bacterial count (Figure 1) (compare the CFU / ml values ​​of Group 3 "titrated particles" with the CFU / ml values ​​of Group 2 "attached fraction"). The bacterial counts obtained after titration were also higher compared to the bacterial counts in the "attached fraction" of the MCC growth composition. 3 The feasibility of removing particles was also supported by cultures of Lactobacillus crispatus and Lactobacillus paracasei (data not shown).

[0300] For Lactobacillus crispatus, Lactobacillus paracasei and Bifidobacterium bifidum, the pH obtained after adding the indicated volume of HCl was 4.0. Tests were performed with Lactobacillus crispatus and Lactobacillus paracasei by adding HCl until the calcium carbonate was completely dissolved (CO2 from the solution). 2 The release of β-lactamase (observed by the release of β-lactamase) resulted in a pH level below 3.0. Such low pH levels reduced bacterial viability by 14-45%. Advantageously, to obtain optimal bacterial viability, the acid should be added in an amount such that bacterial viability is substantially maintained.

[0301] Example 2 MCC-CaCO 3 Enhanced bacterial adhesion to particle mixtures In the following experiments, the effect of the ratio of particle amount to culture medium volume on the level of bacterial attachment was investigated.

[0302] The culture was performed in 50%-50% MCC-CaCO 3Different ratios (up to 200 g particles per L of culture medium) were performed in the presence of particle mixtures (cultures were performed as detailed above).

[0303] Results showed that ratios between 60 g particle mixture per L of culture medium (where calcium carbonate constituted 50% (w / w) of the total weight of the particles) and 200 g particle mixture per L of culture medium enhanced bacterial attachment to the particles (data not shown).

[0304] A composition according to one embodiment of the present invention comprises: (i) a polysaccharide-based material, e.g., MCC; and (ii) a metal carbonate, e.g., CaCO 3 and wherein the total weight of the particles relative to the volume of culture solution can range from 60 g per liter to 250 g per liter, and the metal carbonate can comprise 15% to 85% w / w of the total weight of the particles used, provided, or added in the method.

[0305] Thus, to increase adhesion, it can be advantageous if the ratio between metal carbonate particles and culture liquid used during contact ranges from 9 g per liter to 215 g per liter, taking into account that the advantageous metal carbonate content ranges from 15% to 85% w / w of the total weight of all particles.

[0306] In methods in which the particle further comprises a metal phosphate, e.g. DCP, the metal phosphate and metal carbonate collectively may comprise at least 15% w / w of the total weight of the particle, all weight percentages being based on the dry weight of the particle.

[0307] Typically, using metal carbonate concentrations higher than 85% (w / w of the total weight of the particles) and culturing at the particle:solution ratios specified above results in a viscous culture medium, which requires increased stirring speed to obtain a homogenous culture medium, adversely affecting bacterial adhesion to the particles, and using metal carbonate concentrations lower than 15% (w / w of the total weight of the particles) results in reduced bacterial adhesion.

[0308] The composition can be produced by culturing in the presence of particles having a total weight of less than 60 g per L of solution volume. Under such conditions, the metal carbonate concentration can be 15% to 99% (w / w) based on the total weight of the particles used in the method.

[0309] Compositions prepared according to embodiments of the invention may contain both adherent and non-adherent bacterial fractions, or the two bacterial fractions may be separated, for example, by filtration and / or gravity settling, such as by centrifugation as exemplified herein for performing CFU determinations on each separated bacterial fraction.

[0310] To investigate the effect of consumable calcium on bacterial adhesion to particles, a water-soluble calcium salt (CaCl 2 Different bacterial populations were cultured in the presence of MCC particles in different medium compositions with or without 0.1% calcium ion, to investigate the effect of the presence of consumable calcium on adhesion levels.

[0311] The results showed that the inclusion of calcium ions in the culture medium did not enhance bacterial adhesion to MCC particles (data not shown).

[0312] Taken together, these results indicate that pure MCC or CaCO are superior in achieving increased bacterial attachment to the particles and / or increased total bacterial counts in the composition. 3These results demonstrate the advantage of culturing in the presence of a particle mixture according to the invention compared to culturing in the presence of particles. The results show that the increase in bacterial numbers can be maintained while particles, e.g. CaCO, are added during and / or after the culturing process. 3 The feasibility of at least partially removing particles from bacterial compositions was also demonstrated.

[0313] Example 3 Resistance of different bacterial compositions to external stresses The following examples examine the advantages of compositions prepared according to embodiments of the present invention in withstanding stress conditions, compared to bacterial compositions containing planktonic and / or attached bacteria (96w / p cultures). The bacterial compositions were tested in wet and lyophilized forms. Oxidative stress is a well-accepted assay and was chosen as an exemplary stress condition.

[0314] Figure 2 shows a series of increasing H 2 O 2 Figure 1 shows the survival rate of wet (end of culture) Lactobacillus plantarum cultures after exposure to different concentrations of attached bacterial fractions prepared by different culture methods, namely: (i) CaCO according to an embodiment of the present invention; 3 The resistance of bacteria cultured in the presence of (i) 96w / p MCC particle mixture (1:1 w / w ratio) and (ii) 96w / p was tested. Planktonic growth morphology was used as a reference. 2 O 2 After exposure, the total bacterial counts (CFU) were measured. The results are expressed as a percentage compared to the control for each group (see "H 2 O 2 "Additive-free" was considered to be 100%.

[0315] Figure 3 shows a series of reconstructed and augmented H 2 O 2 The figures show the survival of freeze-dried Lactobacillus plantarum cultures after exposure to concentrations of CaCO 3 The resistance was tested for cultures according to embodiments of the invention in the presence of a 1:1 w / w ratio of H:MCC particles and in suspension growth form. 2 O 2The total bacterial count (CFU) was measured after stress. The results are expressed as a percentage compared to the control for each group (see "H 2 O 2 "Additive-free" was considered to be 100%.

[0316] The results show that cultivation in the presence of the particle mixture according to the invention has clear advantages for withstanding stress conditions compared to other cultivation methods, e.g. methods providing a composition comprising an attached bacterial fraction, and compared to planktonic growth forms.

[0317] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

Claims

1. A composition comprising a population of at least one bacterial strain at least partially attached to particles, wherein the particles comprise (i) a polysaccharide material and (ii) a metal carbonate.

2. The composition according to claim 1, wherein the particles are water-insoluble particles.

3. The composition according to claim 1, wherein the metal carbonate is not produced by the at least one bacterial strain.

4. The composition according to claim 1, having at least one feature selected from (a) comprising particles in the form of a composite material comprising the polysaccharide material and the metal carbonate, and (b) comprising the polysaccharide material and the metal carbonate in different particles.

5. The composition according to claim 1, further comprising the particles dicalcium phosphate (DCP).

6. The composition according to claim 1, further comprising particles lacking a polysaccharide material and a metal carbonate.

7. The composition according to claim 1, wherein the at least one bacterial strain is attached to at least one of the surfaces of the polysaccharide material and the metal carbonate.

8. The composition according to claim 1, wherein the polysaccharide material comprises a cellulose derivative.

9. The composition according to claim 1, wherein the polysaccharide material includes microcrystalline cellulose (MCC).

10. The composition according to claim 1, wherein the metal carbonate contains calcium carbonate.

11. The composition according to claim 1, wherein the metal carbonate is contained in a concentration of 85% (w / w) or less of the total weight of the particles in the composition, based on the dry weight of the particles.

12. The composition according to claim 11, characterized by either (i) containing the metal carbonate at a concentration of at least 15% (w / w) of the total weight of the particles in the composition, based on the dry weight of the particles, or (ii) further containing metal phosphate-containing particles, in which case the metal phosphate and the metal carbonate constitute at least 15% (w / w) of the total weight of the particles in the composition, based on the dry weight of the particles.

13. The composition according to claim 1, further comprising airborne bacteria.

14. The composition according to claim 1, comprising two or more bacterial strains.

15. The composition according to claim 1, provided in a solid form.

16. The composition according to claim 1, further comprising a pharmaceutically acceptable carrier or excipient.

17. The composition according to claim 1, for use in treating or preventing dysbiosis in subjects requiring treatment or prevention of dysbiosis.

18. A method for preparing a composition comprising a population of at least one bacterial strain at least partially attached to particles, wherein the particles comprise (i) a polysaccharide material and (ii) a metal carbonate, and the method is The process of preparing a population of at least one bacterial strain, (i) A step of preparing particles containing a polysaccharide material and (ii) a metal carbonate, A step of bringing the group of at least one bacterial strain into contact with the particles, wherein the contact is performed in a solution, and The step of attaching at least one group of bacterial strains to the particles at least partially. Includes, A method for preparing the composition comprising a population of at least one bacterial strain at least partially attached to the particles.

19. The method according to claim 18, wherein the particles are water-insoluble particles.

20. The method according to claim 18, wherein the contact step includes culturing a population of at least one bacterial strain in a growth medium containing the particles.

21. The method according to claim 18, wherein during the contact step, the particles have at least one feature selected from (a) being in the form of a composite material comprising the polysaccharide material and the metal carbonate, and (b) each of the polysaccharide material and the metal carbonate being contained in different particles.

22. The method according to claim 18, wherein the particles prepared further comprise dicalcium phosphate (DCP).

23. The method according to claim 18, wherein the weight of the metal carbonate relative to the volume of the solution is in the range of 9 g to 215 g per liter, based on the dry weight of the particles.

24. The method according to claim 18, wherein the total weight of the particles prepared relative to the volume of the solution is in the range of 60 g to 250 g per liter, based on the dry weight of the particles.

25. The method according to claim 24, wherein the particles prepared contain the metal carbonate at a concentration of 85% (w / w) or less of the total weight of the particles, based on the dry weight of the particles.

26. The method according to claim 24, characterized in that the particles provided (i) contain the metal carbonate at a concentration of at least 15% (w / w) of the total weight of the particles based on the dry weight of the particles, or (ii) further contain a metal phosphate, in which case the metal phosphate and the metal carbonate constitute at least 15% of the total weight of the particles based on the dry weight of the particles.

27. The method according to claim 18, wherein the metal carbonate is not produced by the at least one bacterial strain.

28. Acid, CO ２ The method according to claim 18, further comprising the step of removing the metal carbonate from the particles by adding or both of the above.

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