Bacterial compositions and methods for growing bacteria on particles

Abstract

The present invention relates inter alia to a composition comprising a population of at least one bacterial strain at least partially attached to a particle comprising (i) a polysaccharide-based material; and (ii) a metal carbonate. Further provided herein are uses of the compositions, for example for the prevention or treatment of dysbiosis, and methods of making the same.

Description

[0001] Cross-references to applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 327,455, filed on April 5, 2022, entitled “Compositions and methods for growing bacteria on particles,” the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention belongs to the field of microbiology, and in particular relates to bacterial compositions in attached and / or aggregated form and methods for their preparation and use. Background Art

[0004] The human body contains a rich and diverse community of microorganisms, forming a network of interactions between bacteria and human cells, which greatly influences our health and even our behavior patterns.

[0005] There is growing evidence supporting the use of probiotics to promote human health, such as by benefiting the immune system and suppressing infection.

[0006] A variety of bacterial compositions have been developed for the prevention or treatment of 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.

[0007] However, one of the challenges of biotherapeutic delivery is the successful survival, colonization, and expansion of the delivered bacteria due to the harsh environmental conditions during entry and arrival at the site of interest.

[0008] Several attempts have been made to develop bacterial compositions that are more suitable for achieving bacterial colonization and providing long-lasting beneficial effects. Each known composition and method has some advantages and disadvantages.

[0009] There remains an unmet need for alternative compositions, such as compositions comprising bacteria attached to alternative particles, that may overcome the shortcomings of existing compositions and provide advantageous methods for preventing or treating diseases and / or conditions.

[0010] Advantageously, such surrogate particles can be at least partially dissolved during the preparation of the composition while maintaining the viability and beneficial effects of the bacteria, thereby enabling the production of a composition comprising a reduced level of inert material and an increased relative level of bacteria in the final product. Summary of the Invention

[0011] 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, wherein the particle comprises: (i) a polysaccharide-based material; and (ii) a metal carbonate.

[0012] In another aspect of the present invention, a method for preparing a composition comprising a population of at least one bacterial strain at least partially attached to particles is provided, wherein the particles comprise: (i) a polysaccharide-based material; and (ii) a metal carbonate, the method comprising the steps of: 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, wherein the contacting is carried out in a solution; and allowing the population of at least one bacterial strain to at least partially attach to the particles; thereby preparing a composition comprising a population of at least one bacterial strain at least partially attached to the particles.

[0013] In some embodiments, the particles are water-insoluble particles.

[0014] In some embodiments, the metal carbonate is not produced by at least one bacterial strain.

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

[0016] In some embodiments, the particles have at least one characteristic selected from the group consisting of: (a) being in the form of a composite comprising a polysaccharide-based material and a metal carbonate; and (b) comprising each of the polysaccharide-based material and the metal carbonate within separate particles.

[0017] 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. In some embodiments, a portion of the particles comprise a polysaccharide-based material and a metal carbonate that are not blended together within the same particle.

[0018] In some embodiments, each particle can include an additional material. In some embodiments, the particle includes at least one additional material.

[0019] In some embodiments, the additional material is or includes dicalcium phosphate (DCP).

[0020] In some embodiments, the particles further comprise dicalcium phosphate (DCP).

[0021] In some embodiments, the compositions and / or methods further comprise particles that are free of polysaccharide-based materials and metal carbonates.

[0022] In some embodiments, the at least one bacterial strain is attached to at least one of: a surface of a polysaccharide-based material; and a surface of a metal carbonate.

[0023] In some embodiments, the particles include multiple types of polysaccharide-based materials. In some embodiments, the particles include multiple types of metal carbonates.

[0024] In some embodiments, the polysaccharide-based material is or includes a cellulose derivative.

[0025] In some embodiments, the polysaccharide-based material is or includes microcrystalline cellulose (MCC).

[0026] In some embodiments, the metal carbonate is or includes calcium carbonate.

[0027] In some embodiments, the composition includes the metal carbonate at a concentration of up to 85% (w / w) of the total weight of the particles in the composition, based on the dry weight of the particles.

[0028] In some embodiments, the composition is characterized by either: (i) comprising a 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 comprising particles comprising a metal phosphate, and wherein 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.

[0029] In some embodiments, the w / w ratio of the polysaccharide-based material to the metal carbonate, based on the dry weight of the particle, is between 1:10 and 10: 1. In some embodiments, the w / w ratio of the polysaccharide-based material to the metal carbonate, based on the dry weight of the particle, is between 1:6 and 6:1.

[0030] In some embodiments, the composition comprises trace amounts of metal carbonates.

[0031] In some embodiments, the composition further comprises bacteria that are not attached to particles. In some embodiments, the composition further comprises planktonic bacteria.

[0032] In some embodiments, the composition comprises two or more bacterial strains.

[0033] In some embodiments, the composition comprises trace amounts of metal carbonates.

[0034] In some embodiments, the composition is provided in solid form.

[0035] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier or excipient.

[0036] In some embodiments, the composition is obtained by a method comprising contacting, mixing, incubating and / or culturing a population of at least one bacterial strain with particles, and allowing the bacteria to at least partially attach to the particles.

[0037] In some embodiments, the composition is obtained by a method comprising culturing a population of at least one bacterial strain in the presence of particles.

[0038] In some embodiments, the present invention provides methods for preparing the compositions disclosed herein.

[0039] In some embodiments, allowing the population of at least one bacterial strain to at least partially attach to the particles comprises culturing the population with the particles.

[0040] In some embodiments, allowing the population of at least one bacterial strain to at least partially attach to the particles comprises incubating the population with the particles for a time sufficient to allow the population to attach to the particles, such as from 1 hour to 15 days.

[0041] In some embodiments, allowing the population of at least one bacterial strain to at least partially attach to the particles comprises culturing the population with the particles until the population attaches to the particles, eg, from 1 hour to 15 days.

[0042] In some embodiments, the method further comprises the step of mixing a population of at least one bacterial strain with the particles in dry form prior to the contacting step.

[0043] In some embodiments, contacting comprises mixing, incubating, and / or culturing. In some embodiments, inoculation is the initial stage of the culturing step and is performed in the presence of particles. In some embodiments, 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, the different steps of the method can be performed in the same or different solutions. In some embodiments, the contacting step comprises culturing a population of at least one bacterial strain in a growth medium containing particles.

[0044] In some embodiments, the method comprises contacting two or more bacterial strains with the particles.

[0045] In some embodiments, at least a portion of the particles used in the methods of the present invention form a composite comprising a polysaccharide-based material; and a metal carbonate. In some embodiments, a portion of the particles used in the methods of the present invention include a polysaccharide-based material and a metal carbonate that are not blended together in the same particle.

[0046] In some embodiments, during the contacting step, the particles have at least one characteristic selected from the group consisting of: (a) being in the form of a complex comprising a polysaccharide-based material and a metal carbonate; and (b) comprising each of the polysaccharide-based material and the metal carbonate within different or separate particles.

[0047] In some embodiments, provided particles include at least one additional material.

[0048] In some embodiments, the weight of the metal carbonate relative to the volume of the solution is in a range of between 9 g / 1 L and 215 g / 1 L based on the dry weight of the particles.

[0049] In some embodiments, the total weight of the particles provided relative to the volume of the solution is in a range of between 60 g / 1 L and 250 g / 1 L based on the dry weight of the particles.

[0050] In some embodiments, provided particles comprise a metal carbonate at a concentration of up to 85% (w / w) of the total weight of the particle based on the dry weight of the particle.

[0051] In some embodiments, provided particles are characterized by either: (i) comprising a metal carbonate at 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, and wherein the metal phosphate and the metal carbonate constitute at least 15% of the total weight of the particle based on the dry weight of the particle.

[0052] In some embodiments, the method further comprises the step of eliminating metal carbonates from the particles by adding acid, CO2, or both.

[0053] In another aspect of the present invention, a method of treating a patient is disclosed, comprising administering a composition according to the present invention to the patient.

[0054] According to another aspect, there is provided a method for preventing or treating dysbiosis in a subject in need thereof, comprising administering to the subject an effective amount of a composition according to the present invention.

[0055] In some embodiments, the present invention provides a method for preparing a composition disclosed herein, comprising the steps of: inoculating a solution with a population comprising at least one bacterial strain; and culturing the population comprising at least one bacterial strain in a solution comprising particles for a time sufficient to allow the bacteria to at least partially attach to the particles, wherein the particles comprise: (i) a cellulose-based material; and (ii) a metal carbonate.

[0056] According to another aspect, there is provided a composition produced according to the inventive method disclosed herein.

[0057] According to another aspect, a pharmaceutical composition is provided, comprising the composition of the invention disclosed herein and an acceptable carrier.

[0058] In some embodiments, the pharmaceutical composition is used to treat or prevent dysbiosis in a subject in need thereof.

[0059] In some embodiments, the composition further comprises an acceptable carrier. In some embodiments, the composition is used to treat or prevent dysbiosis in a subject in need thereof. In some embodiments, the composition is used for agricultural or veterinary purposes.

[0060] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of embodiments of the present invention, exemplary methods and / or materials are described below. In the event of a conflict, the patent specification (including definitions) shall prevail. In addition, materials, methods and examples are illustrative only and are not intended to be necessarily restrictive.

[0061] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given below. However, it should be understood that while indicating preferred embodiments of the present invention, the detailed description and specific examples are given by way of illustration only, as various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art based on this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Included are bar graphs showing bacterial counts (Log CFU / ml) of Bifidobacterium bifidum after incubation in the following conditions: as a planktonic growth form (in the absence of particles); in the presence of particles, the combined particle-attached and non-attached bacterial components (labeled "Group 1"); the particle-attached bacterial component alone (labeled "Group 2"); and after partial elimination of metal carbonate particles from the particle-attached bacterial component by acid, i.e., elimination of metal carbonate particles from the Group 2 composition (labeled "Group 3").

[0063] Figure 2Included are bar graphs showing the survival of wet (at the end of incubation) Lactobacillus plantarum cultures after exposure to increasing H2O2 concentrations. The resistance of attached bacterial compositions prepared by different incubation methods was tested: (i) incubation in the presence of a CaCO3:MCC granular mixture (1:1 w / w ratio) according to embodiments of the present invention; and (ii) bacteria cultured on 96 w / p. Planktonic growth forms were used as a reference. After H2O2 exposure, total bacterial counts (CFU) were measured. Results are expressed as a percentage compared to the control, untreated bacteria of each group ("no H2O2 added" was considered 100%).

[0064] Figure 3 Included is a bar graph showing the survival of freeze-dried Lactobacillus plantarum cultures after reconstitution and exposure to increasing H2O2 concentrations. Resistance was tested in adherent bacterial fractions and planktonic growth forms prepared by culturing in the presence of a CaCO3:MCC particle mixture (1:1 w / w ratio) according to embodiments of the present invention. Following H2O2 stress, total bacterial counts (CFU) were measured. Results are expressed as a percentage compared to the control, untreated bacteria in each group ("no H2O2 added" was considered 100%). DETAILED DESCRIPTION

[0065] According to some embodiments, a composition is provided comprising a population comprising at least one bacterial strain at least partially attached to particles.

[0066] According to some embodiments, there is provided a composition comprising a population comprising at least one bacterial strain grown in the presence of a particle as described herein.

[0067] 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 particles.

[0068] According to some embodiments, methods are provided for obtaining increased bacterial attachment to particles compared to using particles comprising a polysaccharide-based material in the absence of metal carbonate particles, for example, when using particles comprising: (i) a polysaccharide-based material; and (ii) a metal carbonate.

[0069] According to some embodiments, methods are provided for growing a population comprising at least one bacterial strain (e.g., gut-derived bacteria, skin-derived bacteria, vagina-derived bacteria) comprising the steps of mixing, contacting and / or culturing in the presence of particles as described herein.

[0070] 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 a saline solution, a phosphate buffered solution, a growth medium, or the like. In some embodiments, the population of at least one bacterial strain is contacted with the particles while the bacteria and / or particles are in a wet or hydrated form. In some embodiments, the population and particles are initially contacted in a dry or non-hydrated form, such as after a drying process, and then the solution is added and contacted or cultured. 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.

[0071] In some embodiments, solutions include aqueous solutions and liquid solutions.

[0072] In some embodiments, the methods and compositions according to the present invention have several advantageous properties (e.g., increased bacterial attachment to particles, increased bacterial load, and / or increased ability to adhere to mucus surfaces), as shown and described in detail below. In some embodiments, particle-attached bacteria prepared according to the present invention are resistant to environmental stress and persist under adverse conditions in various environments. In some embodiments, particle-attached bacteria prepared according to the present invention are capable of and / or configured to adhere to a surface, e.g., to enable successful bacterial colonization in a subject to which the bacteria are administered.

[0073] In some embodiments, the composition is characterized by an increase in the number of bacteria attached to the particles, e.g., when used or cultured in the presence of particles as described herein, 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 increased bacterial attachment is within the range of a 1.1 to 50-fold increase, 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. Each possibility represents a separate embodiment of the present invention.

[0074] In some embodiments, the terms "bacteria," "polysaccharide-based material," "metal carbonate," and "metal phosphate" as used throughout the specification include both singular and plural references unless the context clearly dictates otherwise.

[0075] In some embodiments, the term "a population of at least one bacterial strain" includes a population of at least one bacterial strain and a population of more than one bacterial strain.

[0076] In some embodiments, with respect to interactions between bacteria and particles, the terms "attachment" and "adhesion" are interchangeable and refer to the adsorption of bacteria to a surface / particle, e.g., through weak interactions and / or stronger interactions, e.g., through flagella, pili, lipopolysaccharides, exopolysaccharides, collagen-binding adhesion proteins, etc.

[0077] In some embodiments, the population of at least one bacterial strain is attached to the particles during culturing. In some embodiments, the population of at least one bacterial strain is grown in the presence of the particles.

[0078] In some embodiments, the present invention provides methods for increasing bacterial load, for example, when used and cultured in the presence of particles as described herein compared to using or culturing in the presence of particles comprising microcrystalline cellulose in the absence of metal carbonate particles and / or compared to planktonic culture in the absence of particles. In some embodiments, the bacterial load increased at the end of culture is in the range of 1.1 to 50 times increased compared to using or culturing in the presence of particles comprising microcrystalline cellulose in the absence of metal carbonate particles and / or compared to planktonic culture, for example, 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 bacterial load, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0079] In some embodiments, the composition according to the present invention comprises CaCO 3 , which advantageously has mucoadhesive properties, e.g., the ability to adhere to and / or remain on mucus surfaces, e.g., through interaction with mucin. In some embodiments, the composition according to the present invention is capable of and / or configured to adhere to mucus surfaces (e.g., skin, intestinal, or vaginal epithelial cells), e.g., to enable successful colonization of bacteria in a subject to which the bacteria are administered. In some embodiments, the composition is configured to adhere to and / or remain on mucus surfaces, and to release bacteria from the composition over a long period of time in the target area. For example, bacteria that are beneficial for skin, vaginal, or intestinal treatments can be included in the composition, and the bacteria can be retained and released in the target area without being washed away to achieve maximum therapeutic effect.

[0080] In some embodiments, the method of the present invention includes a step of partially or completely eliminating / decomposing / dissolving metal carbonate particles. In some embodiments, after the elimination or decomposition step, the survival rate and beneficial effects (such as function) of the bacteria remain substantially unchanged. In some such embodiments, for example, compared with the ratio before the elimination step, the composition comprises an inert material with reduced content and an increased bacterial content. Generally, the term "inert material" refers to a substance that has no biological activity for the intended use of the composition. In some embodiments, substantially maintaining the survival rate, beneficial effects and / or function of the bacteria refers to at least 80% of the initial value before the elimination / decomposition step. In some embodiments, maintaining between 80% and 100% of the initial value before the elimination / decomposition step.

[0081] In some embodiments, bacterial viability is determined by any method known to the skilled artisan, such as, but not limited to, colony forming units (CFU) of viable bacteria, live-dead staining, propidium azidobromide qPCR (PMA-qPCR), metabolic assays, spectrophotometry, or any combination thereof. Methods for determining bacterial viability, such as those disclosed herein, are common and readily apparent to the skilled artisan.

[0082] In some embodiments, beneficial effects are determined by measuring the ability of the bacteria to affect metabolic processes in a subject and / or ameliorate any disease, disorder, or condition in the subject; measuring the potential metabolic routes / pathways of the bacteria, such as the potential to produce, synthesize, consume, and / or utilize certain metabolites / organic compounds.

[0083] In some embodiments, the present invention also provides methods for increasing bacterial resistance and compositions comprising resistant bacteria that can withstand harsh environments and extreme manufacturing conditions, such as antibiotic-resistant, acid-resistant, heat-resistant, and cold-resistant bacteria, for example, compared to bacteria grown in other growth forms.

[0084] In some embodiments, the compositions disclosed herein are administered to a subject in need thereof before, simultaneously with, together with, or after the 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-processed with an antibiotic and / or an acidic substance. In some embodiments, the antibiotic / acidic substance is released or administered before the beneficial / probiotic bacteria, thereby reducing the abundance of pathogens in the target tissue and facilitating the successful colonization of the delivered beneficial or probiotic bacteria. This combination therapy may be advantageous, for example, in preventing or treating female urogenital tract conditions characterized by elevated pH levels and / or increased abundance of pathogenic bacteria. A non-limiting example of such a condition is bacterial vaginosis, in which the administration of lactic acid-producing bacteria prepared according to the present invention with or without antibiotics and / or acidic substances may be beneficial. Thus, in some embodiments, the compositions according to the present invention are used to treat bacterial vaginosis with or without antibiotics and / or acidic substances. The antibiotics and / or acidic substances may be co-administered with the compositions according to the present invention simultaneously, sequentially, or alternatingly. In some embodiments, the antibiotic is any antibiotic used to treat bacterial vaginosis. Non-limiting examples of antibiotics include metronidazole (Flagyl), clindamycin (Cleocin), and metronidazole. In some embodiments, the acidic substance includes a pH regulator. Non-limiting examples of pH regulators according to 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.

[0085] In some embodiments, extreme manufacturing conditions are, for example, temperatures above 37°C, such as above 38°C, above 39°C, above 40°C, above 45°C, above 50°C, within the range of above 37°C to 40°C, above 37°C to 45°C, above 37°C to 50°C, 38°C to 50°C, or within the range of 50°C to 55°C.

[0086] In some embodiments, a composition comprises: a population of at least one bacterial strain at least partially attached to a particle, wherein the particle comprises: (i) a polysaccharide-based material; and (ii) a metal carbonate.

[0087] In some embodiments, the compositions and / or methods include multiple particle types or forms. In some embodiments, for example, during contacting or culturing, the compositions and / or methods include at least one of: (a) particles in the form of a composite comprising a polysaccharide-based material and a metal carbonate; and (b) particles comprising only one of the following: (i) a polysaccharide-based material or (ii) a metal carbonate. By "particles comprising only one of the following: (i) a polysaccharide-based material or (ii) a metal carbonate," it is meant that the composition or method includes particles comprising polysaccharide-based material without metal carbonate and particles comprising metal carbonate without polysaccharide-based material. In some embodiments, "particles comprising only one of the following: (i) a polysaccharide-based material or (ii) a metal carbonate" means that the polysaccharide-based material and the metal carbonate are not bound, processed together, bonded, connected, and linked to each other, for example, 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 "comprising each of said polysaccharide-based material and said metal carbonate within different particles" are interchangeable. In some embodiments, the particles of the method or composition characterized as "comprising each of said polysaccharide-based material and said metal carbonate within different particles" means that the polysaccharide-based material and the metal carbonate are not contained within the same particle, which means that the polysaccharide-based material and the metal carbonate are not bound, processed together, bonded, connected and linked to each other, for example, by processing and / or any chemical interaction. In these embodiments, the particles may comprise only one of: (i) a polysaccharide-based material or (ii) a metal carbonate and optional additional materials, for example, the composition and / or method comprises particles comprising a polysaccharide-based material without a metal carbonate and particles comprising a metal carbonate without a polysaccharide-based material. In some embodiments, the particles comprising a polysaccharide-based material without a metal carbonate; and the particles comprising a metal carbonate without a polysaccharide-based material are in close contact with each other or suspended in a solution during incubation. In some embodiments, each particle type includes at least one additional material. In some embodiments, the composition and / or method further includes additional types of particles. In some embodiments, the composition and / or method further includes other particle types that do not contain polysaccharide-based materials and metal carbonates, such as particles comprising dicalcium phosphate (DCP). Other particle types can be blended or hybrid particles. In some embodiments, the composition and / or method includes a plurality of different or identical composite particles, such as particles comprising polysaccharide-based materials and metal carbonates and / or particles comprising dicalcium phosphate (DCP) and polysaccharide-based materials. In some embodiments, particles primarily consisting of metal carbonates are considered composite particles.

[0088] In some embodiments, the metal carbonate particles are not produced by the at least one bacterial strain. In some embodiments, the compositions and / or methods include a metal carbonate 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 insoluble in water. In some embodiments, the metal carbonate is not produced by a bacterial biofilm. In some embodiments, the metal carbonate is not produced by the bacteria to which the particles of the present invention are attached. 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, for example, it is not a self-produced exopolysaccharide. In some embodiments, the compositions and / or methods include a polysaccharide-based material 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 insoluble in water. In some embodiments, the polysaccharide-based material is not produced by the bacteria to which the particles of the present invention are attached. In some embodiments, the at least one bacterial strain produces the metal carbonate and / or the polysaccharide-based material. In some embodiments, the particles are not produced by sedimentation from culture medium.

[0089] In some embodiments, the method of preparing the composition comprises the steps of: providing a population of at least one bacterial strain; providing particles, wherein the particles comprise: (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 attach to the particles. In some embodiments, during the contacting or culturing step, the suspended particles have a characteristic selected from the following: (a) being in the form of a complex comprising a polysaccharide-based material and a metal carbonate; (b) comprising only one of the following: (i) a polysaccharide-based material or (ii) a metal carbonate; or both (a) and (b). In some embodiments, particles comprising only one of the following: (i) a polysaccharide-based material or (ii) a metal carbonate include: particles comprising a polysaccharide-based material without a metal carbonate and particles comprising a metal carbonate without a polysaccharide-based material.

[0090] As used herein, the term "population of at least one bacterial strain" refers to any integer equal to or greater than 1. 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 bacterial 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 embodiments, the bacteria can be from one or more taxonomic classifications.

[0091] In some embodiments, the population of at least one bacterial strain is derived from or originates from a biological sample. In some embodiments, the population of at least one bacterial strain is derived from or originates 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 comprising a bacterial community artificially created by combining / mixing selected (two or more) bacterial species.

[0092] As used herein, the terms "source," "derived," and conjugates thereof are interchangeable with the term "obtained" and refer to the source from which the bacteria for cultivation / fermentation are obtained.

[0093] In some embodiments, the population of at least one bacterial strain is derived from or is derived from a stool 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 is derived from: a fecal bacterial population, an intestinal 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 is derived 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 is derived from a plant. In some embodiments, the bacterial population is derived from or is derived from more than one bacterial source.

[0094] In some embodiments, the population comprises at least one mutant strain. As used herein, the term "mutant strain" includes: additions, deletions, substitutions, insertions / deletions, inversions, duplications, or any combination thereof, for example, compared to a naturally occurring bacterial strain (such as, but not limited to, a wild-type strain) or a genetic reference strain.

[0095] In some embodiments, the at least one bacterial strain is a probiotic strain. In some embodiments, the term "probiotic" refers to a bacterial strain that, when administered, for example, in sufficient amounts, can provide a health benefit to a host (e.g., a human), for example, by improving or restoring a healthy flora, affecting the metabolic processes of the subject, and / or stimulating the growth of other microorganisms (particularly those with beneficial properties), improving any disease, disorder, and / or condition in the subject.

[0096] In some embodiments, the population of at least one bacterial strain includes Gram-positive bacteria, Gram-negative bacteria, or both. In some embodiments, the population of at least one bacterial strain includes facultative anaerobes, tolerant anaerobes, obligate anaerobes, or any combination thereof. In some embodiments, the population of at least one bacterial strain includes obligate aerobes, microaerophiles, or any combination thereof. In some embodiments, the population of at least one bacterial strain is isolated from a donor sample. In some embodiments, a single isolated colony is selected from a donor sample. In some embodiments, a complete sample is used in the method according to the present invention, and the composition comprises a mixture of bacterial strains. In some embodiments, the population of at least one bacterial strain includes fecal microbiota, vaginal microbiota, for example, a collection of microorganisms derived from colonization in a subject.

[0097] The bacterial strains may be different. In some embodiments, the population of at least one bacterial strain belongs to the family Lactobacillaceae, Bifidobacterium. In some embodiments, the population of at least one bacterial strain includes a bacterial species that produces hydrogen peroxide (H2O2). In some embodiments, the population of at least one bacterial strain includes the ability to colonize epithelial lining tissue.

[0098] In some embodiments, the population of at least one bacterial strain includes a Lactobacillus strain. In some embodiments, the Lactobacillus strain is characterized by or includes the ability to colonize epithelial lining tissue. In some embodiments, the epithelial lining tissue includes vaginal tissue. In some embodiments, the population of at least one bacterial strain includes 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 plantarum, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus salivarius, Lactobacillus delbrueckii, or any combination thereof. In some embodiments, the population of at least one bacterial strain includes at least one bacterium selected from Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus rhamnosus, and any combination thereof. In some embodiments, the population of at least one bacterial strain includes Lactobacillus crispatus. In some embodiments, the population of at least one bacterial strain includes Lactobacillus rhamnosus. In some embodiments, the population of at least one bacterial strain includes Lactobacillus paracasei.

[0099] In some embodiments, the population of at least one bacterial strain includes at least one bacterial strain characterized by or including the ability to colonize skin tissue. In some embodiments, the population of at least one bacterial strain includes at least one of the following species: Lactobacillus plantarum, Propionibacterium acnes, Corynebacterium tuberculostearicum, Propionibacterium acnes, Corynebacterium tuberculostearicum, Corynebacterium tuberculostearicum, Staphylococcus hominis, Staphylococcus epidermidis, Staphylococcus hemolyticus, Streptococcus mitis, Propionibacterium acnes, Corynebacterium tuberculostearicum, 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, Corynebacterium afermentans, Staphylococcus haemolyticus, Micrococcus luteus, Staphylococcus epidermidis, Micrococcus luteus, Corynebacterium aurimucosum, Non-fermenting Corynebacterium, Staphylococcus capitis, Enhydrobacter aerosaccus, Corynebacterium kroppenstedtii, Corynebacterium simulans, Veillonella parvula, Corynebacterium simulans, Corynebacterium amylolyticus amycolatum), Corynebacterium resistens, Lactobacillus pentosus, Lactobacillus rhamnosus, or any combination thereof.In some embodiments, the population of at least one bacterial strain includes Lactobacillus plantarum.

[0100] In some embodiments, the population of at least one bacterial strain includes a Staphylococcus strain, such as Staphylococcus epidermidis. In some embodiments, the population of at least one bacterial strain includes a Bifidobacterium strain, such as Bifidobacterium bifidum. In some embodiments, the population of at least one bacterial strain includes Escherichia coli Nissle 1917 strain.

[0101] In some embodiments, the phrase "at least partially attached to the particles" refers to the following range of bacteria in the composition and / or the total bacteria present at the end of the contacting or culturing step: 0.01% to 100%, 1% to 95%, 5% to 90%, or 10% to 85%, such as at least 0.01% to 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 or range therebetween. In some embodiments, the bacteria are attached to at least one of: (i) a surface of a polysaccharide-based material; (ii) a surface of a metal carbonate material; or both (i) and (ii). In some embodiments, the majority of the attached bacteria adhere to the surface of the metal carbonate particles (e.g., greater than 50% and up to 100%, 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% of the total attached bacteria component, or any value or range therebetween).

[0102] In some embodiments, "bacteria at least partially attached to the particles" includes both direct and indirect attachment or adhesion of bacteria to the particles. In some embodiments, indirect attachment includes attachment to attached bacteria and / or attachment to a matrix formed by attached / adhered bacteria.

[0103] In some embodiments, attachment can be determined by scanning electron microscopy (SEM) techniques; by separating attached and unattached bacterial fractions, for example, by filtration and / or gravity sedimentation, such as centrifugation, and determining bacterial viability in the attached fraction; and / or by any other method known to those skilled in the art. The total bacteria present in the composition and / or in the attached and unattached fractions at the end of the contacting or culturing step can be determined by any method of bacterial viability known to those skilled in the art, as disclosed herein. In some embodiments, the bacteria are separated from the particles prior to determining bacterial viability.

[0104] In some embodiments, bacterial attachment and the percentage of bacterial attachment to total bacteria in the culture system are assessed as follows: the particle-attached bacterial component is separated from the non-attached component by centrifugation (e.g., at 48×g; 21±2° C. for 3 minutes) and the supernatant containing the non-attached bacteria is removed. In some embodiments, the pellet is washed with 0.9% saline, centrifuged repeatedly (e.g., at 48×g; 21±2° C. for 3 minutes), and the supernatant is removed to obtain a pellet of the particle-attached bacterial component. The pellet can be resuspended with 0.9% saline, and the resuspended component can be titrated to assess the bacterial survival rate in the attached component. The percentage of bacterial attachment can be assessed by determining the bacterial survival rate in the total sample before separating the total sample into two components and calculating the bacterial survival rate of the attached component from the survival rate of the entire sample.

[0105] In some embodiments, the term "particle" refers to a substance / material that is suitable, configured or adapted to attach and / or grow at least one bacterial strain. In some embodiments, the particle includes a surface to which bacteria can adhere. In some embodiments, in the context of particles, the term "surface" can be interchanged with the term "surface area" to refer 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 the void (i.e., "empty") space in the material. In some embodiments, the term "porous" includes having an uneven surface area. In some embodiments, the term "particle" refers to discrete three-dimensional shaped materials that are unique and separable from other substances / components added in the method, but this does not exclude that the components are in contact with each other.

[0106] In some embodiments, when the population of at least one bacterial strain is derived from or originates from a biological sample, the particles are not derived from the sample. In some embodiments, when the population of at least one bacterial strain is derived from or originates from a biological sample, the sample does not contain particles disclosed herein.

[0107] As used herein, the terms "particle," "nanoparticle," "microparticle," "nanosphere," and "microsphere" are used interchangeably.

[0108] In some embodiments, all numerical percentages and w / w ratios associated with particles herein are based on the particles in dry form. In some embodiments, the term "dry (dry)" in connection with the percentage and weight characteristics of the particles can be interchanged with the term "non-hydrated". In some embodiments, the term "dry" refers to particles that have undergone a drying process. In some embodiments, the term "dry" refers to particles that have a water content of less than 5%. In some embodiments, "dry particles" refer to particles that do not contain unbound water. In some embodiments, the particles include 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 water content of less than 5% is obtained. In some embodiments, the isolated particles are first subjected to a step of separating attached bacteria, for example by vortexing. The water content can be determined according to Karl Fischer titration, loss on drying (LOD), and / or any other method known to the skilled person.

[0109] 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" means that the particles are substantially insoluble in water, such as saline, phosphate-buffered saline, and growth medium. In some embodiments, the term "substantially insoluble" means that at least 90 wt.% of the particles, preferably 95 wt.% of the particles, are insoluble in water. In some embodiments, the solubility of the particles in water is equal to or less than 2.5 g / 1000 ml at a temperature in the range of 20°C to 60°C, such as 20°C, 25°C, 37°C, 60°C, and / or at a pH in the range of 5.0 to 8.0. Each possibility represents a separate embodiment of the present invention. In some embodiments, the particles used according to the present invention are insoluble in water at physiological pH. In some embodiments, the particles used according to the present invention are insoluble in water at a pH in the range of about 1.0 to 8.0.

[0110] In some embodiments, compositions and / or methods include various types of particles, for example, particles with different size distributions. In some embodiments, the diameter range of the particles is 0.1 micron to 1 centimeter. In some embodiments, the diameter range of the particles is 5 microns to 1 cm. In some embodiments, the diameter range of the particles is 0.1 to 500 microns. In some embodiments, the range of the particles is 1 micron to 50 millimeters. In some embodiments, compositions and / or methods include particles with a diameter less than 1 micron. In some embodiments, compositions and / or methods include particles in the range of 30 to 500 microns (for example, 40 to 400 microns, 50 to 300 microns, 60 to 200 microns, 70 to 100 microns or any value and range therebetween). In some embodiments, the compositions and / or methods comprise 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 or range therebetween), or any value or range therebetween. In some embodiments, the particles have a diameter of at least 0.2 microns, at least 2 microns, at least 3 microns, at least 5 microns, at least 10 microns, at least 15 microns, at least 20 microns, at least 30 microns, at least 40 microns, at least 50 microns, at least 60 microns, at least 70 microns, at least 80 microns, at least 90 microns, at least 100 microns, at least 200 microns, at least 300 microns, at least 400 microns, at least 500 microns, at least 600 microns, at least 700 microns, at least 800 microns, at least 900 microns, at least 1 centimeter in diameter, or any value or range therebetween. Each possibility represents a separate embodiment of the present invention.

[0111] In some embodiments, the particles have an average diameter in the range of 0.2 to 1500 microns. In some embodiments, the particles have an average diameter in the range of 50 to 1200 microns, 50 to 1100 microns, 50 to 1000 microns, 55 to 1200 microns, 55 to 1000 microns, 57 to 1200 microns, or 60 to 1000 microns, including any ranges therebetween. Each possibility represents a separate embodiment of the present invention.

[0112] 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.

[0113] In some embodiments, the term "particle" includes any particle, e.g., in any shape within the size range implemented. In some embodiments, the particle can be round, amorphous, irregular, spherical, ellipsoidal, flower-shaped, cubic, spherical, elongated, rod-shaped, have any other shape, or any combination thereof.

[0114] In some embodiments, at least a portion of the particles comprise a composite or mixture comprising: (i) a polysaccharide-based material; and (ii) a metal carbonate. In some embodiments, during the contacting and / or culturing step, the particles are in the form of a composite comprising: a polysaccharide-based material; and a metal carbonate. In some embodiments, the composite particles are formed during the contacting and / or culturing step, for example, by chemical interaction. In some embodiments, the particles are provided to the method as a composite or mixture comprising: (i) a polysaccharide-based material; and (ii) a metal carbonate.

[0115] In some embodiments, the term "at least a portion of the particles comprises a composite or mixture comprising: (i) a polysaccharide-based material; and (ii) a metal carbonate" refers to a range of between 1% and 100%, between 5% and 90%, between 10% and 80%, or between 20% and 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), or any value and range therebetween, of the particles being in the form of composite particles comprising: a polysaccharide-based material; and a metal carbonate. All percentages are from the total particles present in the composition and / or provided, added, or used in the method. In some embodiments, the form of the remaining particles comprises only one of the following: a polysaccharide-based material or a metal carbonate. In some embodiments, the "composite particles" and / or "remaining particles" include additional types of materials.

[0116] As used herein, the term "composite" or "mixture" refers to a particle composed of different materials. In some embodiments, the composite particle comprises: (i) a polysaccharide-based material; and (ii) a metal carbonate and optionally additional materials or substances.

[0117] In some embodiments, a portion of the particles comprises only one of the following: a polysaccharide-based material or a metal carbonate. In some embodiments, the term "a portion" in relation to particles comprising only one of the following: a polysaccharide-based material or a metal carbonate refers to the remaining percentage of particles provided, added or used in the composition and / or method.

[0118] In some embodiments, during the contacting and / or culturing steps, the particles are isolated particles that contain only one of the following: a polysaccharide-based material or a metal carbonate. In some embodiments, the polysaccharide-based material; and the metal carbonate are provided, added or used in the method as unique particles, each particle containing only one of the polysaccharide-based material or the metal carbonate. In some embodiments, the term "unique" includes the terms "separate," "separate," "different," or "non-blended," and refers to the composition of the particles relative to the polysaccharide-based material and the metal carbonate. In some embodiments, the "unique particles" are blended or mixed with additional materials. In some embodiments, the unique particles form a complex during and / or after contact with the solution.

[0119] In some embodiments, the particles in the composition and / or particles provided, added or used in the method contain only one of the following: less than 100% polysaccharide-based material or metal carbonate; for example, 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 value and range therebetween. All percentages are from the total particles in the composition and / or particles provided, added or used in the method.

[0120] In some embodiments, a majority (e.g., greater than 50%, such as between 51% and 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 value or range therebetween) of the particles provided, added, or used in the composition and / or method are composite particles comprising a combination of at least one polysaccharide-based material; and a metal carbonate. All percentages are from the total particles in the composition and / or method.

[0121] In some embodiments, the compositions and / or methods include multiple particle types, e.g., comprising 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.

[0122] In some embodiments, each particle comprises an additional material, thereby forming a "blended or mixed particle". In some embodiments, the additional material comprises dicalcium phosphate (DCP). In some embodiments, each particle comprises one material. In some embodiments, each particle comprises two or more different materials. In some embodiments, the composition and / or method further comprises particles that do not contain polysaccharide-based materials and metal carbonates. In some embodiments, the composition and / or method comprises particles that comprise DCP, polysaccharide-based materials, or a combination thereof.

[0123] In some embodiments, a material is uniformly dispersed within a single particle. The term "uniformly" means that the material is substantially evenly dispersed throughout the single particle, e.g., not present at a significantly higher concentration in any part of the particle. In some embodiments, different regions of the particle have substantially the same material composition. In some embodiments, different regions of the particle have different material compositions.

[0124] In some embodiments, the composite particles comprising a polysaccharide-based material and a metal carbonate have at least one characteristic selected from the group consisting of: (i) not physically processed; (ii) physically processed; (iii) not a co-processed mixture; (iv) a co-processed mixture; and (v) the polysaccharide-based material and the metal carbonate are bonded, connected, or linked by chemical interactions.

[0125] In some embodiments, the compositions and / or methods include blended or mixed particles, e.g., a mixture comprising various materials having at least one characteristic selected from the group consisting of: (i) not physically processed; (ii) physically processed; (iii) not a co-processed mixture; (iv) a co-processed mixture; and (v) the materials comprising the particles are bonded, linked, or associated through chemical interactions.

[0126] In some embodiments, the compositions and / or methods include a plurality of composite, blended, or mixed particles comprising any combination of features (i) to (v).

[0127] In some embodiments, physical processing includes compression, shear forces, etc. In some embodiments, particles in the form of a co-processed mixture include materials / substances that are combined by the application of mechanical pressure.

[0128] In some embodiments, the composite, blended or mixed particles are added as separate materials in the process and form composite, blended and mixed particles, for example, through any chemical bonds, forces and / or interactions.

[0129] In some embodiments, the method further includes the step of 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 the 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 includes the step of mixing the solutions containing the dispersed polysaccharide-based material and the metal carbonate, for example, by simple blending. In some embodiments, simple blending includes conventional mixing or blending operations such as shaking, stirring, etc. In some embodiments, the composite particles containing the polysaccharide-based material and the metal carbonate are formed by dispersing two separate particle types in a solution and / or by mixing / combining two separate dispersed solutions, each containing a different particle type / material. Non-limiting examples of solutions include saline solutions, phosphate buffered solutions, growth media, etc. In some embodiments, dispersion is performed before, simultaneously with, and / or after inoculating or adding the bacteria to the solution. In some embodiments, after blending, the solution is a uniform mixture of the 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 additional particle types, are dispersed in the solution simultaneously, one after the other, or both. In some embodiments, the particles / materials are dispersed in the solution in dry form (e.g., powder form), in wet form, or both.

[0130] In some embodiments, at least a portion of the particles are blended, mixed or composite particles that contain a mixture of more than one substance. In some embodiments, a portion of the particles include a polysaccharide-based material; and a mixture of metal carbonates. In some embodiments, the composition and / or method includes particles comprising a polysaccharide-based material. In some embodiments, the composition and / or method includes particles comprising a metal carbonate. In some embodiments, the composition and / or method includes particles comprising only one of the following: a polysaccharide-based material or a metal carbonate, and optional additional materials. In some embodiments, the composition and / or method includes particles comprising a polysaccharide-based material and substantially no metal carbonate. In some embodiments, the composition and / or method further includes particles comprising a metal carbonate and substantially no polysaccharide-based material. In some embodiments, the composition and / or method includes particles consisting primarily 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) to (iii).

[0131] In some embodiments, the weight ratio between: (i) particles comprising a polysaccharide-based material and being substantially free of metal carbonates: (ii) particles comprising a metal carbonate and being substantially free of polysaccharide-based material: and (iii) composite particles comprising a polysaccharide-based material and a metal carbonate is in a range of 1:1:1 to 10:10:10, for example 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 weight / weight / weight (w / w / w) based on dry granular form.

[0132] In some embodiments, "polysaccharide-based material" refers to a material comprising a polysaccharide, while the term "polysaccharide" refers to a pure carbohydrate, such as starch or cellulose. In some embodiments, "polysaccharide-based material" refers to a naturally occurring polymer composed of glucose units, for example linked by 1-4 beta glycosidic bonds. In some embodiments, the polysaccharide-based material is a linear polysaccharide, for example, a material that forms a crystalline or partially crystalline structure, for example, at a temperature in the range of 20 to 60° C., for example, at a temperature selected from 20, 25, 37, 60° C. and / or at a pH in the range of 5 to 8. In some embodiments, the term "polysaccharide-based material" includes oligosaccharide-based materials.

[0133] In some embodiments, the polysaccharide-based material comprises a material / substance based on cellulose; pectin; inulin; dextrin; maltodextrin; glycogen; starch, such as starch derived from corn or potato; amylose; alginate; chitosan, such as hydrophobically modified chitosan (HMC); fructooligosaccharides (FOS); galacto-oligosaccharides (GOS); or any combination thereof.

[0134] In some embodiments, the polysaccharide-based material comprises fructooligosaccharides (FOS); galacto-oligosaccharides (GOS) or a combination thereof. In some embodiments, the cellulose-based material comprises a material comprising cellulose fibers, such as microcrystalline cellulose (MCC).

[0135] 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 an electric charge. As used herein, the term "net charge" refers to the total charge of a material. For example, a material with a positive net charge is a material with an excess of protons. In one embodiment, where the composition and / or method comprises multiple types of polysaccharide-based materials, each type of polysaccharide-based material can 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.

[0136] In some embodiments, the polysaccharide-based material is negatively charged at a pH value in the range of 3.5 to 10.0, for example, MCC is negatively charged.

[0137] In some embodiments, "metal carbonate" is interchangeable with the term "mineral salt" and refers to a mineral salt comprising a metal cation and a carbonate anion (CO3 -2 Typically, metal carbonates can decompose, for example, when reacting with an acid, to produce salt, water, and carbon dioxide gas (CO2).

[0138] 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 various types of metal carbonates including various types of metal cations. In some embodiments, the metal cations include monovalent, divalent, trivalent metal cations, or any combination thereof.

[0139] In some embodiments, the metal carbonate is or includes a divalent cation. In some embodiments, the metal cation is or includes calcium (Ca 2+ ), barium (Ba 2+ ), manganese (Mn 2+ ), iron (Fe 2+ ), magnesium (Mg 2+ ), zinc (Zn 2+ ) or any combination thereof. In some embodiments, the metal cation comprises calcium (Ca 2+ ).

[0140] In some embodiments, the metal cation is or includes a calcium cation (Ca 2+ ), the metal carbonate is or includes calcium carbonate (CaCO 3 ). In some embodiments, the polysaccharide-based material is or includes a cellulose derivative, such as microcrystalline cellulose (MCC). In some embodiments, at least a portion of the particles in the composition and / or method are in the form of composite particles comprising a mixture of: (i) MCC; and (ii) calcium carbonate. In some embodiments, a portion of the particles in the composition and / or method comprise (i) MCC; and (ii) calcium carbonate as separate, non-blended particles. In some embodiments, each particle further comprises additional materials.

[0141] "Calcium carbonate" (CaCO3) that can be used according to the present invention can be synthesized or manufactured by any method known in the art, for example to obtain nanometer and micrometer (micron) materials, and is generally available in several forms, shapes, and crystal structures. In some embodiments, the forms and types used according to 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.

[0142] In some embodiments, the particles comprising metal carbonates, such as calcium carbonate, are precipitated particles. In some embodiments, the metal carbonates used according to the present invention are pre-prepared particles, such as those available on the market. In some embodiments, the precipitated particles are formed in a container for contacting bacteria with the particles. In some embodiments, the method further comprises a step of precipitating (e.g., dripping) calcium oxide into a solution, the solution being, for example, contained in a culture container; thereby providing particles comprising calcium carbonate. In some embodiments, the method further comprises a precipitation step, such as dripping dry Na2CO3 and CaCl2 into a solution and / or mixing a solution of Na2CO3 and CaCl2, thereby providing 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 synthesized calcium carbonate. In some embodiments, the particles comprising calcium carbonate are ground calcium carbonate. In some embodiments, calcium carbonate contains impurities.

[0143] In some embodiments, the average diameter of the calcium carbonate particles is in the range of 0.2 to 100 microns, for example 2-20 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 comprises amorphous CaCO , crystalline CaCO , or any combination thereof. In some embodiments, the calcium carbonate comprises a crystalline phase selected from calcite (β-CaCO ), aragonite (λ-CaCO ), vaterite (μ-CaCO ); and any combination thereof.

[0144] In some embodiments, the metal carbonate and the polysaccharide-based material are present during the contacting and / or incubation period at concentrations that will allow the population to at least partially attach to the particles.

[0145] Without being bound by a particular theory or mechanism, using high metal carbonate concentrations may result in a viscous culture medium that may require increased stirring speed to obtain a homogenous culture medium, thereby negatively impacting bacterial attachment to the particles; and using low metal carbonate concentrations will also result in lower bacterial attachment.

[0146] Thus, in some embodiments, during the contacting and / or culturing step, the total weight of the particles relative to the volume of the solution (e.g., growth medium) is up to 250 g per liter. In some embodiments, during the contacting and / or culturing step, the total weight of the particles relative to the volume of the solution (e.g., growth medium) is at least 60 g per liter. In some embodiments, during the contacting and / or culturing step, the total weight of the particles relative to the volume of the solution (e.g., growth medium) is in a range of between 60 g per liter and 250 g per liter, such as between 60 g per liter and 200 g per liter.

[0147] In some embodiments, the metal carbonate comprises up to 85% w / w of the total weight of the particles at this particle:solution ratio. In some embodiments, the metal carbonate comprises up to 85% w / w of the total weight of the particles at this particle:solution ratio. In some embodiments, the metal carbonate comprises up to 75% w / w of the total weight of the particles at this particle:solution ratio. In some embodiments, the metal carbonate comprises up to 75% w / w of the total weight of the particles at this particle:solution ratio. In some embodiments, the metal carbonate comprises at least 15% w / w of the total weight of the particles at this particle:solution ratio. In some embodiments, the metal carbonate comprises at least 25% w / w of the total weight of the particles at this particle:solution ratio. In some embodiments, the metal carbonate content ranges from 15% w / w to 85% w / w of the total weight of the particles at this particle:solution ratio. In some embodiments, the metal carbonate content ranges from 25% w / w to 75% w / w of the total weight of the particles at this particle:solution ratio. All percentages are w / w of the total weight of the particles provided, added or used in the method. Thus, in some embodiments, the metal carbonate constitutes at least 15% w / w of the total weight of the particles in the composition. In some embodiments, the metal carbonate constitutes at least 25% w / w of the total weight of the particles in the composition. In some embodiments, the metal carbonate constitutes up to 85% w / w of the total weight of the particles in the composition. In some embodiments, the metal carbonate is up to 85% w / w of the total weight of the particles in the composition. In some embodiments, the metal carbonate constitutes up to 75% w / w of the total weight of the particles in the composition. In some embodiments, the metal carbonate is up to 75% w / w of the total weight of the particles in the composition. In some embodiments, the metal carbonate content ranges from 25% w / w to 75% w / w of the total weight of the particles in the composition. In some embodiments, the metal carbonate content ranges from 15% w / w to 85% w / w of the total weight of the particles in the composition.

[0148] In some embodiments, the metal carbonate content of the particles provided, added or used in the composition and / or in the method ranges from 15% to 85% (w / w), 15% to 80% (w / w), 15% to 75% (w / w), 15% to 70% (w / w), 15% to 65% (w / w), 15% to 6 ... to 55% (w / w), 15% to 50% (w / w), 15% to 45% (w / w), 15% to 40% (w / w), 20% to 85% (w / w), 25% to 75% (w / w), 25% to 85% (w / w), 25% to 80% (w / w), 20% to 50% (w / w), 20% to 48% (w / w), 20% to 45% (w / w) or 20% to 40% (w / w).

[0149] In some embodiments, the remaining percentage is particles comprising polysaccharide-based material. In some embodiments, the polysaccharide-based material is present in an amount ranging from 15% to 85% (w / w), 15% to 80% (w / w), 15% to 75% (w / w), 15% to 70% (w / w), 15% to 65% (w / w), 15% to 60% (w / w), 15% to 55% (w / w), 15% to 100% (w / w), 15% to 150 ... % to 50% (w / w), 15% to 45% (w / w), 15% to 40% (w / w), 20% to 85% (w / w), 25% to 75% (w / w), 25% to 85% (w / w), 25% to 80% (w / w), 20% to 50% (w / w), 20% to 48% (w / w), 20% to 45% (w / w) or 20% to 40% (w / w).

[0150] In some embodiments, the weight of the metal carbonate particles provided relative to the volume of the solution is at least 9 g per 1 L. In some embodiments, the weight of the metal carbonate particles provided relative to the volume of the solution is up to 215 g per 1 L. In some embodiments, the weight of the metal carbonate particles provided relative to the volume of the solution ranges from 9 g per 1 L to 215 g per 1 L, from 9 g per 1 L to 200 g per 1 L, or from 15 g per 1 L to 190 g per 1 L.

[0151] In some embodiments, the weight of the polysaccharide-based material particles provided relative to the volume of the solution is at least 9 g per 1 L. In some embodiments, the weight of the polysaccharide-based material particles provided relative to the volume of the solution is up to 215 g per 1 L. In some embodiments, the weight of the polysaccharide-based material particles provided relative to the volume of the solution is in the range of 9 g per 1 L to 215 g per 1 L, 9 g per 1 L to 200 g per 1 L, or 15 g per 1 L to 190 g per 1 L.

[0152] In some embodiments, during the contacting or culturing step, the polysaccharide-based material, such as MCC; and the metal carbonate, such as calcium carbonate, are present in the composition and / or solution in a weight ratio (w / w) 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 ratio (w / w) 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.

[0153] 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.

[0154] In some embodiments, when the particle further comprises a metal phosphate, the metal phosphate and the metal carbonate together constitute at least 15% (w / w) of the total weight of the particle. In some embodiments, the ratio between the metal phosphate and the 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 or range therebetween. Each possibility represents a separate embodiment of the present invention. In some embodiments, when the particle further comprises a metal phosphate, the metal phosphate and the metal carbonate together constitute at least 25% (w / w) of the total weight of the particle. In some embodiments, the ratio between the metal phosphate and the 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 or range therebetween. Each possibility represents a separate embodiment of the present invention.

[0155] In some embodiments, when the total weight of the particles relative to the volume of the solution (e.g., growth medium) during the contacting and / or culturing step is less than 60 g / 1 L, the concentration of the metal carbonate may be greater than 85% w / w of the total weight of the particles. In such embodiments, the metal carbonate concentration may be up to 99% w / w of the total weight of the particles. In other embodiments, the metal carbonate concentration is at least 15% w / w of the total weight of the particles. In other embodiments, 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 between 15% w / w and 99% w / w of the total weight of the particles, such as between 15% w / w and 95% w / w, between 15% w / w and 90% w / w, between 20% w / w and 95% w / w, between 25% w / w and 99% w / w, between 25% w / w and 90% w / w, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention. All percentages are w / w based on the total weight of particles provided, added or used in the process.

[0156] Thus, in some embodiments, the metal carbonate constitutes at least 15% w / w of the total weight of the particles in the composition. In some embodiments, the metal carbonate constitutes at least 25% w / w of the total weight of the particles in the composition. In some embodiments, the metal carbonate constitutes 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% w / w to 99% w / w of the total weight of the particles in the composition, such as from 15% w / w to 95% w / w, from 15% w / w to 90% w / w, from 20% w / w to 95% w / w, from 25% w / w to 99% w / w, from 25% w / w to 90% w / w, or any value or range therebetween. Each possibility represents a separate embodiment of the present invention.

[0157] In some embodiments, CaCO 3 is at least partially entrapped and / or adsorbed by the polysaccharide-based material.

[0158] In some embodiments, the metal carbonate (eg, CaCO 3 ) and the polysaccharide-based material are connected, adsorbed, fused, or surface-associated with each other, for example, through any chemical bond, force, and / or interaction.

[0159] In some embodiments, the method includes contacting a population of at least one bacterial strain with a particle; and allowing the population of at least one bacterial strain to at least partially attach to the particle.

[0160] The word "contacting" is used herein in its broadest sense and refers to any type of binding action, for example, bringing bacteria into proximity with a particle, enabling the bacteria to attach / adhere to a particle. In some embodiments, contacting comprises incubating, growing or culturing the bacteria together with or in the presence of the particle, for example, as opposed to planktonic culturing in the absence of particles. In some embodiments, contacting comprises combining the bacteria, particles, and solution in any order, combination, and / or subcombination, including pre-mixing two elements before adding a third element, or mixing all elements simultaneously. In some embodiments, the method comprises the steps of inoculating the bacteria in the solution and then culturing the bacteria in the solution containing the particles. In some embodiments, the method comprises the steps of providing the solution containing the particles as a previous step and then adding a population of at least one bacterial strain. In some embodiments, the contacting and culturing are performed simultaneously or as subsequent steps.

[0161] In some embodiments, the contacting or culturing is performed in a solution. In some embodiments, the solution is selected from a buffer, saline, phosphate buffered saline, and a growth medium. In some embodiments, the contacting comprises culturing the bacteria in the presence of particles.

[0162] In some embodiments, the term "culture" includes the term "fermentation", and refers to the maintenance, propagation and / or growth of cells (such as bacterial cells) in vitro on or in various buffers and / or culture media under laboratory or industrial conditions. In some embodiments, the cells propagated or grown may be not identical with the parental cells in morphology, gene or phenotype. The term "fermentation" has its ordinary meaning in this area. In some embodiments, the term "fermentation" is used to refer to a microbial metabolic process in this article, which includes using, for example, bacteria to convert sugar into acid and / or gas.

[0163] Suitable culture media will be apparent to those skilled in the art and can be any known or commercially available culture media, or can be synthesized and / or custom designed, for example, to support the maintenance, proliferation, and / or growth of cultured cells. Non-limiting examples of culture media include, but are not limited to, tryptic soy broth (TSB), yeast casein fatty acid (YCFA), Robertson cooked meat, fortified Clostridium medium (RCM) broth, nutrient broth, fastidious anaerobic broth (FAB), lysogenic broth (LB), 1 / 4LB, heart infusion broth, Wilkins-Chalgren broth, Brucella broth (Brucella broth), wheat bran, brain heart infusion (BHI), brain heart infusion agar (BHI1), M9 minimal medium, or 0.2X BHI, Gifu anaerobic medium (GAM), gut microbiota medium (GMM), Columbia blood agar (CBA), chocolate agar (CHOC), tryptic soy agar (TSY), fastidious anaerobic bacteria agar (FAA), tryptic yeast extract dextrose (TYG), cooked meat agar (BEEF), Bifidobacterium selection medium (BSM), phenylethyl alcohol agar (PEA), actinomycetes isolation agar (AIA), colistin naladixic acid agar (CNA), McKay agar (MK), mannitol salt agar (MSA), deMan Rogosa Sharpe agar (MRS), Bacteroides bile esculin agar (BES), In some embodiments, the culture medium comprises pharmaceutical grade ingredients. In some embodiments, the culture medium comprises food grade ingredients. In some embodiments, the culture medium comprises components suitable for veterinary use.

[0164] In some embodiments, the method includes the steps of providing bacteria contained in a vessel, and contacting or culturing the bacteria in the vessel. In some embodiments, the term "vessel" refers to any receptacle in which bacteria can be cultured in conventional fermentation techniques, such as a bioreactor, a flask, a test tube, a microtiter dish, a well plate, a multiwell plate assembly, a petri dish, etc.

[0165] In some embodiments, the method further comprises the step of inoculating the solution with a population of at least one bacterial strain, e.g., as a prior step to the contacting step. In some embodiments, the contacting comprises the step of inoculating the solution with a population of at least one bacterial strain. In some embodiments, the contacting comprises the step of incubating the population of at least one bacterial strain in the solution comprising the particles for a period of time sufficient to allow the at least one bacterial strain to at least partially attach to the particles. In some embodiments, the contacting step comprises culturing the population of at least one bacterial strain, e.g., in a growth medium. In some embodiments, the contacting comprises inoculating the solution comprising the particles with bacteria; and incubating or culturing the bacteria with the particles for a period of time sufficient to allow the bacteria to at least partially attach to the particles.

[0166] In some embodiments, contact or culture is performed under conditions that allow at least one bacterial strain population to at least partially adhere to the particles. In some embodiments, suitable conditions include a pH level, for example, in the range of 5 to 8, under flow, shaking, stirring, agitation, static, moist, low humidity, or any combination thereof. In some embodiments, suitable conditions include selecting a culture medium that supports the growth of the cultured bacterial strain. In some embodiments, suitable conditions are to contact or culture the at least one bacterial strain population with the particles for a period of time ranging from 1 hour to 15 days. In some embodiments, the time range sufficient to allow at least partial attachment / adhesion of bacteria to the particles is: 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 present invention.

[0167] In some embodiments, contact or culture is carried out under conditions comprising: static, flowing, stirring, shaking, agitation or any combination thereof, for example, at 50 to 750 revolutions per minute (RPM), 50 to 650 RPM, 100 to 750 RPM, 100 to 700 RPM, 150 to 700 RPM, 200 to 750 RPM, 130 to 690 RPM, 90 to 720 RPM, 70 to 550 RPM, 110 to 710 RPM or any value and scope therebetween. Each possibility represents a separate embodiment of the present invention. In some embodiments, contact is carried out under static conditions. In some embodiments, contact is carried out under static conditions and then contacted under flowing conditions. In some embodiments, contact is carried out under flowing conditions and then contacted under static conditions.

[0168] In some embodiments, the bacteria are contacted with the particles or cultured under shear conditions. In some embodiments, the solution (e.g., growth medium) applies shear forces to the bacteria during the contacting or culture step. In some embodiments, the term "shear force" refers to the movement of the solution relative to the bacteria, e.g., relative to the bacteria attached to the particles. In some embodiments, shear conditions include contacting or culturing the bacteria under mixing, shaking, flowing, rolling, vortexing, stirring, rocking, agitating, bubbling, pumping, bubble formation, or any combination thereof.

[0169] In some embodiments, the terms "flow," "stirring," "shaking," and "agitation" refer to conditions that cause the liquid phase within the culture vessel to move or be in motion. In some embodiments, the movement is axial, radial, mixing, distributed, or any combination thereof. The movement or motion can be achieved by using a mechanical device, such as an impeller, a moving platform, a rocker, a vibrator; manually; automatically, or any combination thereof. In some embodiments, the term "static conditions" refers to conditions in which the liquid phase is not subjected to agitation or any other motion action (manual, automatic, and / or mechanical).

[0170] In some embodiments, the contacting or culturing step is included in culturing under anaerobic conditions. In some embodiments, the term "anaerobic conditions" refers to a condition in which free oxygen is lower than 500ppm, 450ppm, 400ppm, 350ppm, 300ppm, 250ppm, 200ppm, 150ppm, 100ppm, 50ppm or 10ppm or any value and scope therebetween. Each possible embodiment represents a separate embodiment of the present invention. In some embodiments, anaerobic conditions include conditions in which free oxygen is not provided. In some embodiments, anaerobic conditions include conditions in which free oxygen is lacking.

[0171] In some embodiments, the contacting or culturing step comprises culturing under aerobic conditions. In some embodiments, the term "aerobic conditions" refers to conditions comprising the presence of molecular oxygen. In some embodiments, the oxygen concentration is greater than 20% (v / v of the total gas present during the culture).

[0172] In some embodiments, the growth medium further comprises elements that enhance bacterial attachment to the particles.

[0173] In some embodiments, after bacteria attach to the particles, some or all of the bacteria may be separated, and / or other bacteria in the suspension may attach to the particles, the bacteria attached / adhered to the particles, and / or the matrix formed by the attached / adhered bacteria.

[0174] In some embodiments, the method includes a step of separating attached and non-attached bacterial components, for example, producing a composition comprising attached bacteria; and a composition comprising non-attached bacteria. In some embodiments, the method further includes a step of removing bacteria that are not attached to the particles from a solution (e.g., a culture medium or growth medium). In some embodiments, the separation or removal step is performed at at least one time point selected from: before, during, after the incubation, contacting or culturing step, and any combination thereof. In some embodiments, the non-attached / non-adherent bacteria are or include planktonic bacteria. In some embodiments, the non-attached bacteria do not contain sessile bacteria.

[0175] In some embodiments, separation of components or removal of non-attached bacteria is performed by, or includes, filtration and / or gravity sedimentation, such as centrifugation. In some embodiments, the term "filtration" includes all separation techniques and any other process that utilizes filters that can separate components.

[0176] In some embodiments, the composition comprises bacteria attached to particles and / or in planktonic form. In some embodiments, the composition comprises bacteria attached to particles. In some embodiments, the composition comprises bacteria in planktonic form.

[0177] In some embodiments, the method comprises the step of: eliminating metal carbonates from the particles, particle-attached bacterial components, and / or from the solution in which the contacting or incubation is conducted; thereby producing a composition comprising trace amounts of metal carbonates.

[0178] In some embodiments, the step of eliminating metal carbonate is performed at least at one time point during the preparation method. In some embodiments, the elimination step is performed at a time point selected from the following: (i) during contacting, such as during culturing; (ii) at the end of the contacting step, such as at the end of culturing; (iii) before separating the particle-attached bacteria from the non-attached bacterial component; (iv) after separating the particle-attached bacteria from the non-attached bacterial component; or (v) at any time point in combination of (i) to (iv).

[0179] In some embodiments, the elimination step is performed within the vessel in which the contacting occurs and / or in a separate vessel. In some embodiments, the elimination step is performed in a pellet containing bacteria. In some embodiments, the elimination step is performed in a pellet containing bacteria. In some such embodiments, the pellet can be resuspended, for example, in saline, prior to elimination.

[0180] In some embodiments, the term "elimination of metal carbonates" includes partial or complete decomposition of metal carbonates, e.g., depending on the amount of acid and / or CO added to the solution during the contacting step, e.g., during the incubation step. In some embodiments, between 1% and 100% (based on weight or moles) of the initial calcium carbonate particles are eliminated. 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% of the initial calcium carbonate particles are eliminated, or any value and range therebetween.

[0181] In some embodiments, where the particles comprise a metal phosphate, such as DCP, the addition of acid and / or CO2 may result in partial or complete elimination of the DCP from the particles, the bacterial attachment components, and / or from the solution in which the contacting or incubation occurs.

[0182] In some embodiments, the elimination comprises adding an acid to the solution. In this embodiment, carbon dioxide gas (CO 2 ), a metal salt, and water are produced. For example, in one embodiment, wherein: (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 released by the solution and leaves the container in which contact is made. In another embodiment, wherein: (i) magnesium carbonate (MgCO 3 ) is a metal carbonate compound, and (ii) the acid is HCl→ carbon dioxide, magnesium chloride (MgCl 2 ) and water are produced. In some embodiments, carbon dioxide gas is released by the solution and leaves the container in which contact is made.

[0183] 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 is water-soluble at the temperature at which the contacting is performed.

[0184] In some embodiments, after elimination of the metal carbonates, free metal ions are removed from the solution by adsorption, absorption, chelation, or any combination thereof.

[0185] In some embodiments, the term "acid" as used herein refers to and Lowry's definition. 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 astatide (HAt), hydrogen tennesside (HTs); or any combination thereof. In some embodiments, the acid comprises HCl, HBr, and HI.

[0186] In some embodiments, eliminating comprises creating a CO2-rich environment in the solution in which the contacting occurs. In such embodiments, wherein calcium carbonate (CaCO3) is the metal carbonate compound, the calcium carbonate reacts with carbon dioxide and water to produce calcium bicarbonate [(Ca(HCO3)2].

[0187] In some embodiments, eliminating comprises contacting the composition with an effective amount of a chelator or chelating agent.

[0188] In some embodiments, after the elimination / decomposition step, the composition, particles, and / or solution in which the contacting occurs comprises trace amounts of metal carbonates. In some embodiments, during and / or after the elimination step, an increase in the level of any decomposition product can be detected by any method known to the skilled artisan, such as measuring salt concentration, carbon dioxide, and / or calcium bicarbonate levels in the container or solution in which the contacting occurs.

[0189] In some embodiments, the composition comprises trace amounts of metal carbonate. In some embodiments, the term "trace amount" refers to a concentration in the range of 0.01% to less than about 5%. In some embodiments, the term "trace amount" refers to a concentration of metal carbonate less than about 5% by weight of the total weight of the composition, such as less than 4%, less than 3%, less than 2%, less than 1%, or any value and range therebetween. In some embodiments, the term "trace amount" refers to a concentration of metal carbonate less than about 1% by weight of the total weight of the composition.

[0190] In some embodiments, partial or complete dissolution of the metal carbonate particles, for example by acid and / or CO2, can be achieved by calculating the number of moles of acid and / or CO2 added to the solution relative to the number of moles of metal ions present in the particles, as known to those skilled in the art. In some embodiments, complete dissolution of the metal carbonate particles can be achieved by adding acid to the solution in a molar ratio equal to the number of moles of metal ions present in the particles. In one embodiment, wherein: (i) calcium carbonate (CaCO3) is the metal carbonate compound, (ii) the acid is HCl, and (iii) complete dissolution is desired, the molar ratio between HCl and calcium carbonate can be 2:1. In some embodiments, the molar ratio between HCl and calcium carbonate is lower, for example, 1.5:1 or 1.25:1 or less. In some embodiments, the amount of acid added does not affect the viability of the bacteria, for example, the pH level during the dissolution process is suitable to enable the bacteria to proliferate, grow and / or be maintained.

[0191] In some embodiments, the composition further comprises bacteria in aggregated form, for example, after decomposition of the metal carbonate particles. In some such embodiments, a majority of the attached bacteria adhere to the surface of the polysaccharide-based material (e.g., greater than 50% of the total attached bacterial component, 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 or range therebetween). The total attached bacteria can be determined by separating the two components as described above.

[0192] In some embodiments, the term "bacteria in aggregated form" is interchangeable with the term "bacterial clumps" and refers to the collection of individual bacterial particles into a single body. In some embodiments, the composition according to the present invention comprises an increased content of aggregated bacteria, for example, 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 or range therebetween, compared to a composition 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.

[0193] In some embodiments, the composition comprises bacteria in the form of a biofilm. In some embodiments, the biofilm is in the form of a dried biofilm or a solid form, such as a powder. In some embodiments, the term "biofilm" refers to a bacterial community embedded in a matrix comprising self-produced exopolysaccharides, such as those adhered to the surface of particles. In some embodiments, planktonic bacteria are entrapped, embedded, integrated, or embedded beneath, upon, or within the biofilm.

[0194] In some embodiments, the bacterial load of the composition; or at the end of the contacting or culturing step, is at least 1·E 4 , for example, at least 1·E 6 Per 1g, for example, per 1g of particles provided, added or used in the method according to the present invention; the total weight of bacteria at the end of each culture and / or the total weight of each composition. In some embodiments, the method further comprises the step of harvesting the cultured bacteria. In some embodiments, the bacterial load of the harvested cultured bacteria is at least 1·E 4 , for example, at least 1·E 6 Per 1 g, for example per 1 g of particles and / or per total weight of harvested bacteria.

[0195] In some embodiments, the bacterial load ranges from at least 1·E 4 To at least 1·E 20 , at least 1·E 5 To at least 1·E 15 , at least 1·E 5 To at least 1·E 10 or at least 1·E 7 To at least 1·E 9 In some embodiments, the bacterial load is at least 1·E 6 , 1·E 7 , 1·E 8 , 1·E 9 , 1·E 10 , 1·E 11 , 1·E 12 , 1·E 14 , 1·E 16 , 1·E 20 or any value and range therebetween. Each possibility represents a separate embodiment of the present invention. In some such embodiments, the bacterial load can be per 1 g, for example, per 1 g of particles provided, added, or used in the method, per the total weight of the cultured bacteria at the end of 1 g of the composition, and / or per 1 g of the composition.

[0196] In some embodiments, bacterial load determination is performed according to any standard method available and known to those skilled in the art or ordinary skill in the art, non-limiting examples of which include but are not limited to plate counts, spectrophotometric (turbidimetric) analysis, or other methods.

[0197] In some embodiments, bacterial load is determined by viable bacterial colony forming units (CFU), quantitative polymerase chain reaction (qPCR), flow cytometry, live-dead staining, propidium azide bromide qPCR (PMA-qPCR), microscopy, metabolic assays, other commonly used methods, or any combination thereof.

[0198] 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 the group consisting of fungi, bacteriophages, viruses, archaea, and the like, or any combination thereof, e.g., which are derived from or are found in, constitute, or are known to exist in the subject's environmental niche.

[0199] In some embodiments, the particles are dispersed within the composition or within a vehicle comprising the composition. In some embodiments, the term "dispersed" with respect to the presence of particles in a composition or vehicle includes particles that are substantially present throughout the composition or vehicle and are not present at a higher concentration in any portion of the composition or vehicle. Additionally, the term "dispersed" also includes particles that are present in a localized area of ​​the composition or vehicle.

[0200] In some embodiments, the compositions and / or methods according to the present invention comprise 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, together as a co-culture; or a mixture of the two. The bacterial strains and / or the ratios between the different strains may vary, for example, depending on the intended use. In some embodiments, the strains and ratios can be selected based on the desired characteristics of the strains, such as the ability to lower the pH level in the target area, inhibit the growth of specific pathogens, etc. In some embodiments, the different strains are isolated / derived from different sources, such as different donors. In some embodiments, when the bacteria are derived from a sample, such as a donor, the composition and / or culture method can be enriched with additional bacteria or supplemented with additional bacteria.

[0201] According to some embodiments, there is provided a composition produced according to the method of the present invention.

[0202] According to some embodiments, a pharmaceutical composition is provided, comprising a composition disclosed herein and an acceptable carrier or excipient.

[0203] In some embodiments, the term "pharmaceutical composition" encompasses the terms "dietary composition" and "nutritional composition."

[0204] In some embodiments, the terms "dietary composition," "nutritional composition," and "nutraceutical composition" refer to a composition suitable for consumption as a food supplement, e.g., to supplement a normal diet, correct a nutritional deficiency, maintain an adequate intake of certain nutrients, and / or support specific physiological functions.

[0205] In some embodiments, the composition according to the present invention is administered to the subject immediately after cultivation. In some embodiments, the composition is administered after storage (e.g., at room temperature, at a temperature within the range of 2-8 ° C or below -18 ° C). In some embodiments, the composition is provided in a solid form, such as lyophilized, spray-dried, or frozen state. In some embodiments, solid compositions disclosed herein are stable for at least three months at room temperature (e.g., at a temperature selected from about 20, 21, 22, 23, 24, and 25 ° C). In this context, the term "solid" refers to the physical state of the material. In some embodiments, the composition is a solid composition. In some embodiments, the composition is a lyophilized composition.

[0206] In general, the terms "lyophilization" and "freeze drying" are interchangeable and refer to the steps of 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 long periods 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 vehicle, for example for use as a suppository, or can be reconstituted by adding a semi-liquid or liquid solution. The volume added during the reconstitution process can be similar, lower, or higher than the initial volume of the solution before the lyophilization process.

[0207] In some embodiments, the composition is for pharmaceutical use. In some embodiments, the composition is for agricultural use. In some embodiments, the composition is for veterinary use. In some embodiments, the composition is used as an over-the-counter medication, for example, as a supplement.

[0208] In some embodiments, the pharmaceutical composition is used to treat or prevent dysbiosis in a subject in need thereof. In some embodiments, the compositions disclosed herein are used to prepare a medicament for treating or preventing a dysbiosis-related condition.

[0209] In some embodiments, the composition is a synthetic composition. In some embodiments, the term "synthetic composition" refers to a composition comprising bacteria grown or cultured in vitro. In some embodiments, the synthetic composition includes an artificial composition. In some embodiments, the synthetic composition is a man-made composition, such as, but not limited to, a composition prepared or produced in a laboratory and / or manufacturing site or facility. In some embodiments, the synthetic composition does not include a composition 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 comprises: a cryoprotectant, a lyoprotectant, an antioxidant, or any combination thereof. In some embodiments, the composition comprises at least one metabolite produced in vitro by at least one bacterium.

[0210] In some embodiments, a method for preventing or treating dysbiosis in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of a composition according to the present invention.

[0211] The term "therapeutically effective amount" or "effective amount" refers to the amount required to prevent, improve and / or treat a disease, disorder or condition. The effective dose can vary depending on the sex, age and weight of the subject, the disease or condition and its severity, and any other factors that will be recognized by those skilled in the art.

[0212] The compositions and formulations disclosed herein can be used both internally and externally, for example, to treat or prevent dysbiosis. In some embodiments, the compositions and formulations can be applied to the surface of a body part of a subject in need thereof.

[0213] As used herein, the term "surface of a body part of a subject" refers to external body surfaces visible to the naked eye, such as the skin of the face, throat, scalp, chest, back, ears, neck, hands, elbows, knees, and other skin parts; and surfaces of internal body parts, for example, which are part of the internal anatomy of a subject, such as, but not limited to, the oral cavity, gastrointestinal tract, and lower reproductive tract, including but not limited to the vagina.

[0214] As used herein, the term "dysbiosis" is characterized by an alteration, imbalance, impairment, and / or dysfunction of the microbial community of 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 deep within the skin, that is typically associated with a disease, health condition, or clinical condition. This imbalance may exist in any microbial community, including but not limited to imbalances in the gastrointestinal tract, skin, oral cavity, bronchial tubes, vagina, or rectum.

[0215] In some embodiments, the dysbiosis is 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 and polycystic ovary syndrome. In some embodiments, the dysbiosis is vaginal and female genital dysbiosis, such as that associated with bacterial vaginosis, urinary tract infections, human papillomavirus infections, urogenital tract infections, urinary tract infections, candidal infections, infertility, sexually transmitted infections, and gynecological cancers. 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.

[0216] In some embodiments, dysbiosis comprises an imbalance in the microbial flora. In some embodiments, the pharmaceutical composition is used to regulate the flora in a subject in need thereof. In some embodiments, the compositions disclosed herein are used to manufacture a medicament for regulating the flora. In some embodiments, a method for regulating the flora in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of a composition according to the present invention. In some embodiments, the microbial flora comprises vaginal flora, intestinal flora, or skin flora.

[0217] In some embodiments, the term "modulating the microbiome" includes inhibiting or reducing the abundance of bacteria that are typically associated with a disease, health condition, or clinical symptoms; restoring a healthy microbiome balance; increasing diversity and / or achieving colonization of beneficial bacteria on any tissue and / or any body part of an organism, including but not limited to any external or internal surface of the body and deep skin that is typically associated with a disease, health condition, or clinical symptoms.

[0218] In some embodiments, the term "treating dysbiosis" includes generating a desired / predetermined microbial profile in a subject's environmental niche that is beneficial, for example, to improve the subject's responsiveness to an administered drug.

[0219] In some embodiments, a "microbial profile" includes bacterial diversity (eg, alpha diversity or beta diversity); relative abundance of bacteria; and / or bacterial load.

[0220] In some embodiments, the terms "bacterial load," "bacterial viability," and "bacterial count" are interchangeable. In some embodiments, the composition comprises viable bacteria and the bacterial count is measured by CFU.

[0221] In some embodiments, the bacterial load in a composition produced according to the present invention is calculated per gram of dry form particles introduced into a culture medium or container. In some embodiments, the bacterial load is calculated per gram of dry particles used in contact with the bacteria. In some embodiments, the weight of the particles added to the culture system is taken into account when measuring the bacterial load of a composition. In some embodiments, the weight of the particles in the final composition is taken into account when measuring the bacterial load of bacteria in a composition.

[0222] In some embodiments, the altered microbiota comprises pathogenic microorganisms. In some embodiments, the altered microbiota is associated with a medical condition and / or is detrimental to the health of a subject (e.g., a human subject).

[0223] As used herein, the term "altered microbiota" refers to a bacterial flora that has deviated from a homoeostatic microbiota, such as in a non-healthy subject.

[0224] 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 can be male or female.

[0225] In some embodiments, the subject in need of treatment has, or is at risk of developing, a disease, disorder, or condition, as described herein.

[0226] In some embodiments, the composition comprises 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 adjuvant, or a simple sterile aqueous solution, such as saline. In some embodiments, the term "pharmaceutically acceptable carrier" refers to any diluent or vehicle suitable for use by humans or other animals. Some examples of materials that can be used as pharmaceutically 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; tragacanth powder; malt, gelatin, talc; excipients such as cocoa butter, 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 glycerol, 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; ethanol and phosphate buffer solutions and other nontoxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of materials that can be used as carriers herein include sugars, starches, cellulose and its derivatives, tragacanth powder, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffered solution, cocoa butter (suppository base), emulsifiers (such as carbomer, hydroxypropyl cellulose, sodium lauryl sulfate) and other non-toxic pharmaceutically compatible substances 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 regard, suitable pharmaceutically acceptable carriers, excipients, and diluents are well known to those skilled in the art, as disclosed, for example, in The Merck Index, 13th edition, Budavari et al., eds., Merck & Co., Inc., Rahway, NJ (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, 10th edition (2004); and the "Inactive Ingredient Guide", US Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, all of which are incorporated herein by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers, and diluents that can be used in the present compositions 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 ed., Gilman et al., eds., Pergamon Press (1990); Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated herein by reference in its entirety. The presently described compositions may also be contained 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 presently described peptides are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol.A variety of methods can be used to prepare liposomes, for example, as reviewed by Coligan, JE et al., Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, and see also U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369. The carriers may comprise a total of about 0.1% to about 99.99999% by weight of the pharmaceutical compositions described herein.

[0227] The pharmaceutical composition can take any physical form required for correct application. The composition can be applied in any suitable form, including but not limited to liquid form, gel form, semi-liquid (e.g., liquid, such as a viscous liquid, containing some solid) form, semi-solid (solid, containing some liquid) form, or solid form. The composition can be provided in, for example, tablet form, pessary form, cream form, suppository form, capsule form, liquid form, food form, chewable form, non-chewable form, oral form, sublingual form, sustained release form, non-sustainable release form, sustained release form, or non-sustained release form.

[0228] In some embodiments, the composition is formulated for administration by a route selected from the group consisting of rectal, parenteral, mucosal, vaginal, nasal, local, topical, pulmonary, ocular, oral, buccal administration, and any combination thereof. In some embodiments, the composition is administered orally. In some embodiments, the composition is administered topically (e.g., by insertion into the vagina of a suppository, capsule, or any other suitable form).

[0229] In some embodiments, the compositions can be placed in various drug delivery systems, including but not limited to capsules, suppositories, dissolvable shells, and the like.

[0230] In some embodiments, the composition is provided in the form of a liquid or semisolid, cream, suppository, pessary, tablet, pill, caplet, capsule, granules, ointment, lotion, gel, spray, sachet, drops, oral solution, suspension, syrup, emulsifier, oil, or film.

[0231] In some embodiments, the composition comprises: a carrier; and at least a cultured bacterial species disposed within the carrier. In some embodiments, the composition comprises a mixture of dried bacteria. In some embodiments, each bacterium is cultured separately. In some embodiments, the composition comprises co-cultured bacteria.

[0232] In some embodiments, the composition is placed within an enteric coating.

[0233] In some embodiments, the composition is configured for pH-dependent targeted release of bacterial strains in the gastrointestinal tract. In some embodiments, the composition is coated with a compound, such as alginate, and configured to release bacteria at a pH found in the intestine of a subject.

[0234] General Description

[0235] It is assumed herein that all numerical values ​​are modified by the term "about." The term "about" generally refers to a range of numbers that one skilled in the art would consider equivalent to the stated value (e.g., having the same function or result). In many cases, when a numerical value is preceded by the term "about," the term "about" is meant to represent ±10%.

[0236] The terms "comprises," "comprising," "includes," "including," "having" and variations thereof mean "including but not limited to."

[0237] The term "consisting of" means "including and limited to . . . . ."

[0238] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if such additional ingredients, steps and / or parts do not materially change the basic and novel characteristics of the claimed composition, method and structure.

[0239] The word “exemplary” is used herein to mean “serving as an example, instance, or illustration.” Any implementation described as “exemplary” is not necessarily to be construed as preferred or advantageous over other implementations and / or to exclude features from other implementations.

[0240] The word "optional" is used herein to mean "is provided in some embodiments and not provided in other embodiments." Unless such features are incompatible, any particular embodiment of the present invention may include a number of "optional" features.

[0241] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The term "or" as used in this specification and the appended claims generally includes "and / or" in its sense unless the content clearly dictates otherwise.

[0242] Throughout this application, various embodiments of the present invention may be presented in 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. Therefore, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0243] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integer) within the indicated range. The phrases "a range between" a first indicated numeral and a second indicated numeral and "a range from" a first indicated numeral to" a second indicated numeral are used interchangeably herein and are intended to include the first and second indicated numerals and all fractions and integers therebetween. For example, a description of "a range between 15% and 85%" should be deemed to include the first and second indicated percentages and all numbers within the indicated range. The phrases "at least," "up to," and "at most" as used herein are intended to include the indicated numerals. For example, "up to 85%" should be deemed to include the indicated numerals.

[0244] As used herein, the term "method" refers to manners, means, techniques and procedures for accomplishing a given task, including but not limited to those known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry, microbiology and medicine, or readily developable from known manners, means, techniques and procedures.

[0245] It should be understood that certain features of the invention described in the context of separate embodiments for the sake of clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for the sake of brevity may also be provided separately, or in any suitable subcombination, or as appropriate in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments unless the embodiment would not function without those elements.

[0246] The description of various embodiments of the present invention has been provided for the purpose of illustration and is not intended to be exhaustive or limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, practical applications, or technical improvements over technologies found in the marketplace, or to enable those skilled in the art to understand the embodiments disclosed herein.

[0247] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the appended claims section find experimental support in the following examples.

[0248] Other terms used herein are meant to be defined by their commonly understood meanings in the art.

[0249] By studying the following examples, those skilled in the art will understand other objects, advantages and novel features of the present invention, which are not intended to be limiting. In addition, each of the various embodiments and aspects of the present invention as described above and claimed in the appended claims is experimentally supported in the following examples.

[0250] Example

[0251] Generally, the nomenclature used herein and the laboratory procedures used in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. These techniques are fully explained in the literature. For example, see "Molecular Cloning: A laboratory Manual" Sambrook et al. (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds.) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methods as described in U.S. Patent Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, JE, ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), 3rd ed.; "Current Protocols in Immunology", Volumes I-III Coligan JE, ed. (1994); Stites et al. (eds.), "Basic and Clinical Immunology" (8th ed.), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds.), "Selected Methods in Cellular Immunology", WH Freeman and Co., New York (1980); available immunoassay methods are widely described 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 Synthesis" Gait, MJ, ed. (1984); "Nucleic Acid Hybridization" Hames, BD, and Higgins SJ, ed. (1985); "Transciption and Translation" Hames, BD, and Higgins SJ, ed. (1984); "Animal Cell Culture" Freshney, RI, ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "APractical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated herein by reference. Other general references are provided in this document.

[0252] In the following examples, various bacterial strains with different characteristics (e.g., different taxonomic groups and different origins) were cultured as embodiments, as described in Table 1 below, to illustrate the superiority of the methods according to the present invention in producing a composition comprising a population of at least one bacterial strain at least partially attached to the particles when using the particles described herein, compared to other culture methods. In the examples, MCC and CaCO3 were used as embodiments of the polysaccharide-based material and metal carbonate particles.

[0253] Materials and methods

[0254] Test bacteria

[0255] Table 1: Tested bacteria.

[0256]

[0257] Particles used

[0258] Microcrystalline cellulose (MCC); and CaCO3 (CAS#: 471-34-1) were used. All weights and ratios of the particles in the Examples section refer to the particles in dry, non-hydrated form. The water content in the particles is less than 5%. The diameter of the particles can range from 0.1 to 500 microns.

[0259] Culture conditions and preparation of bacterial compositions

[0260] Cultivation was performed as described in the "Experimental Procedures" below in sterile bottles containing 200 ml of growth medium with or without 40 g of particles of: (i) MCC; or (ii) MCC and CaCO3 at different ratios (w / w).

[0261] The tested ratios of MCC and CaCO3 were: 75%:25% (30 g:10 g); 50%:50% (20 g:20 g); and 25%:75% (10 g:30 g).

[0262] 100% CaCO3 or 100% MCC particles were also tested.

[0263] Alternatively, a particle mixture comprising MCC / CaCO3 / DCP may be used.

[0264] The culture medium used included: for Lactobacillus crispatus and Lactobacillus paracasei - yeast extract; peptone; Tween; L-cysteine; sodium, potassium, manganese and magnesium salts; and + (D)-glucose. For Bifidobacterium bifidum - Bifidobacterium broth supplemented with L-cysteine; Tween; and + (D)-glucose.

[0265] Planktonic growth (cultivation in the absence of particles) was also performed under the corresponding conditions specified for each bacterium.

[0266] If culturing is done in the presence of particles, pre-mix the particles with the culture medium (adjust the pH of the medium according to the bacteria used - see conditions below) before mixing with the inoculum.

[0267] Experimental procedures

[0268] Cultures of each bacterium were prepared from an initial inoculum stored at -80°C (the inoculum stock contained planktonic bacteria and a 50% solution of glucose in a ratio of 5:1 w / v).

[0269] Fifty (50) μl (for L. crispatus and L. paracasei) or 100 μl (for B. bifidum) inoculum was mixed with 200 ml of culture medium with or without 40 g of particles (100% MCC; 100% CaCO3 or MCC / CaCO3 mixture as specified above) and incubated / cultivated for 10 hours (for L. crispatus and L. paracasei) or 20 hours (for B. bifidum) under the following conditions: pH in the range of ~5.0-6.5; under anaerobic conditions (gas mixture: N2-90%, H2-5%, CO2-5%) at a temperature of 37°C and stirring at about 25 RPM using a magnet during the entire cultivation period.

[0270] In an embodiment, CaCO 3 particles are removed from the bacterial composition by adding an acid solution.

[0271] To quantitatively assess bacterial attachment to the different test particles, the total bacterial counts [expressed as Log(CFU / ml)] were evaluated as follows:

[0272] - At the end of the culture: (i) planktonic growth, i.e., culture without particles ( Figure 1 , “planktonic”); and (ii) cultured in the presence of particles (for total bacterial mass, i.e., combining particle-attached and non-attached bacterial fractions; Figure 1 ). Planktonic culture was used as a reference group; and

[0273] - Bacterial components attached to isolated particles (in Figure 1 The separation steps for each test group were performed as detailed below.

[0274] Comparison of bacterial counts in the different attachment fractions (present in Group 2) provides an indication of any changes in attachment levels when using the particle mixture according to the invention versus incubation in the presence of MCC without CaCO3 particles.

[0275] To assess the feasibility of using acid to dissolve particles, the bacterial components attached to the particles (e.g. Figure 1 Bacterial counts (CFU) were assessed after elimination / titration of CaCO3 particles in the culture medium ("Group 3" in the original text).

[0276] Figure 1 The results in Figure 5 show the bacterial counts (Log CFU / ml) obtained in planktonic cultures of Groups 1-3 and B. bifidum.

[0277] All methods were performed as follows.

[0278] Bacterial load measurement (colony forming units, CFU)

[0279] Unless otherwise stated, CFU measurements were performed by taking a sample from each culture flask and transferring it into three sterile tubes (3 tubes per culture condition; 10 ml per tube).

[0280] For Group 1, high-speed vortexing was performed for 2 minutes to remove bacteria bound to the particles. To assess attachment, CFU measurements were performed from each test condition by serial dilution (titration test), and the bacteria were plated on agar plates in triplicate. Prior to CFU counting, the plates were incubated at 37°C under anaerobic conditions (gas mixture: N2-90%, H2-5%, CO2-5%) for 24-48 hours.

[0281] For Group 2, the particle-attached bacterial fraction was separated from the non-attached fraction by centrifugation (48 × g, 21 ± 2°C for 3 minutes). The supernatant was then removed and the pellet containing the particle-attached bacterial fraction was washed 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 titrated as described in detail for Group 1.

[0282] For Group 3, the particle-attached bacterial fraction was separated from the non-attached fraction by centrifugation (48 × g, 21 ± 2°C for 3 minutes). The supernatant was then removed and the pellet containing the particle-attached bacterial fraction was washed 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 CaCO particles were removed with HCl as detailed below. The titration test was then performed as detailed for Group 1.

[0283] For planktonic growth, aggregates were dissociated / dissociated by high speed vortexing for 2 minutes and titration assays were performed.

[0284] Use acid to dissolve / eliminate CaCO3 particles

[0285] For Lactobacillus crispatus, Lactobacillus paracasei, and Bifidobacterium bifidum, 5M HCl was gradually added to 100-200 μl portions of the saline (0.9%) resuspended fractions from Group 3, prepared as described in detail above, while manually mixing the samples. The exact volumes added to each culture condition are shown in Table 2.

[0286] Typically, HCl is added taking into account the following two criteria:

[0287] 1- The release of CO2 from the solution, indicating that the CaCO3 particle dissolution reaction is occurring; and

[0288] 2- The pH level throughout the dissolution step to ensure bacterial survival and functionality at the end of the CaCO3 pellet dissolution.

[0289] Several HCl volumes were tested, and the volumes listed in Table 2 represent the maximum CaCO3 particle dissolution with the least effect on bacterial counts (bacteria were counted for each added volume, Figure 1 Bacterial counts of Bifidobacterium bifidum for the volumes indicated in the table are shown).

[0290] Table 2: Volume of HCl added according to the different particle ratios tested.

[0291]

[0292] Resistance of attached bacterial components to H2O2 stress

[0293] The advantages of the compositions comprising particle-attached bacteria prepared according to the present invention in withstanding H2O2 stress conditions compared to planktonic bacterial compositions and / or bacterial compositions comprising attached bacteria (96 w / p culture) were examined.

[0294] Bacterial resistance to H2O2 was tested on several compositions: (i) a composition prepared according to an embodiment of the present invention [cultured in the presence of MCC:CaCO3 at a ratio of 1:1 (w / w)]; (ii) a bacterial composition prepared in 96-well plates (labeled "96 w / p culture"), and (iii) a planktonic bacterial composition (labeled "planktonic"). Compositions (i) and (iii) were tested in wet form (at the end of the culture) and after lyophilization. Composition (iii) prepared in 96 w / p was examined in wet form. To measure stress after lyophilization, the dried composition was reconstituted in PBS at room temperature (about 25°C) for 15 minutes. Both the wet and lyophilized forms were processed in the same manner as detailed below.

[0295] Resistance experiments were conducted against Lactobacillus plantarum. Initial bacterial counts were similar under all tested conditions.

[0296] 96w / p culture steps

[0297] In order to compare the advantages of the composition according to an embodiment of the present invention in withstanding stress, a 96 w / p culture was used, in which bacterial growth was carried out in an attached form.

[0298] A 96 w / p culture of Lactobacillus plantarum was prepared from an initial inoculum stored at -80°C [the inoculum stock contained planktonic bacteria in a 5% (v / v) DMSO solution].

[0299] Initially, 50 μl of 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 / cultivated at 37°C under anaerobic conditions (gas mixture: N2-90%, H2-5%, CO2-5%) for 10 hours.

[0300] After incubation for 10 h, measure the OD 600 , dilute the bacterial population to an initial OD of 600 The mixture was incubated at 0.01 and shaken at 100 RPM for another 12 h under the same conditions.

[0301] After incubation for 12 h, measure the OD 600 , dilute the bacterial population to an initial OD of 600 The new bacterial stock was transferred to 96 w / p in triplicate (100 μl per well) and incubated for approximately 24 hours under the same conditions as described in detail above for the static conditions.

[0302] After incubation, the spent solution was aspirated and the culture was gently washed with PBS to remove the planktonic bacterial fraction (not attached to the 96w / p). The supernatant was aspirated from the plate. In the next step, the plate-attached bacterial population was exposed to increasing concentrations of H2O2 by adding medium containing H2O2 as described in detail below.

[0303] Cultivating bacteria in a fermenter according to an embodiment of the present invention

[0304] Cultivation was performed in a 5-liter fermentor containing 2 L of growth medium and in the presence of 400 g of a pellet mixture [MCC and CaCO3 at a 1:1 (w / w) ratio (200 g:200 g)] as described in detail below: "Fermentation step." Prior to adding the inoculum to the fermentor, the pellets were premixed with the medium (the pH of the medium was adjusted to the culture conditions - see below).

[0305] The culture medium includes yeast extract, peptone, Tween, L-cysteine, sodium, potassium, manganese, and magnesium salts, and (D)-glucose.

[0306] Oxidative stress is a well-established assay and was chosen as an exemplary stress condition to examine the advantages of the compositions comprising particle-attached bacteria prepared according to the present invention.

[0307] Fermentation steps

[0308] Lactobacillus plantarum culture was prepared from an initial inoculum stored at -80°C (the inoculum stock contained concentrated planktonic bacteria and glucose 50% solution in a ratio of 5:1 w / v).

[0309] One hundred (100) μl of inoculum was mixed with 300 ml of culture medium and incubated for 10 hours under the following conditions: pH value in the range of ~5.0-6.5; under anaerobic conditions (gas mixture: N2-90%, H2-5%, CO2-5%), temperature 37°C, without stirring.

[0310] Fifty (50) ml of culture was inoculated into a fermenter containing 2 L of medium and 400 g of the pellet mixture. Fermentation / cultivation was carried out for 15 hours with constant stirring. The initial pH was set to 6.9, the final pH was set to 5.94, and the temperature was set to 37°C.

[0311] Under the corresponding conditions, planktonic growth was performed in the absence of particles.

[0312] In preparation for stress measurements of the compositions of the present invention, after incubation, the particle-attached fraction was separated from the unattached fraction (e.g., the float phase) by centrifugation (48 × g; 3 minutes at 21 ± 2°C), the supernatant was removed, and an additional PBS wash and centrifugation (4248 × g; 5 minutes at 21 ± 2°C) were performed. The supernatant was removed and the pellet was subjected to H2O2 stress.

[0313] In preparation for stress measurements of planktonic cultures, cultures were centrifuged, spent medium removed, and then subjected to additional washes with PBS and additional centrifugation (both at 4248 xg; 21 ± 2°C for 5 minutes) prior to stress application.

[0314] H2O2 stress

[0315] The bacteria were subjected to different concentrations of H2O2: 0.1, 0.2 or 0.5% for 1 minute. Different concentrations of H2O2 were prepared in Lactobacillus plantarum culture medium.

[0316] To prevent H2O2-induced stress, 0.2% sodium thiosulfate in PBS was added to the culture.

[0317] To quantitatively assess bacterial survival after stress, bacteria grown at 96 w / p were separated from the well surface by vigorous pipetting; bacteria grown in the presence of MCC:CaCO3 were separated from the pellet by vortexing (high speed for 1.5 minutes). Planktonic cultures were also vortexed. Total bacterial counts were expressed as colony-forming units (CFU) using serial dilutions in PBS and plating on MRS plates. Results are expressed as a percentage compared to the control for each group ("no H2O2 added" was considered 100%).

[0318] Example 1

[0319] Cultivation of bacterial populations in the presence of MCC and CaCO3 particles

[0320] The following examples examine attachment levels during cultivation using a mixture of MCC and CaCO particles. To this end, cultivation in the presence of the mixture was compared with cultivation in the presence of MCC or CaCO particles alone. Planktonic cultivation without particles served as a reference in these experiments.

[0321] Three different bacteria were cultured separately: Bifidobacterium bifidum, Lactobacillus paracasei, and Lactobacillus crispatus. As detailed above in the "Culture Conditions and Preparation of Bacterial Compositions" and "Experimental Procedures" sections, different ratios of particle mixtures and pure MCC or pure CaCO particles were used to culture the different bacteria. After cultivation, the attachment levels of the different test groups were measured and compared.

[0322] Figure 1 Shown are bacterial counts (CFU / ml) of Bifidobacterium bifidum grown in the absence of particles (planktonic growth form; labeled "planktonic"); in the presence of particles, the combined particle-attached and non-attached bacterial fraction (labeled "Group 1"); the particle-attached bacterial fraction alone (labeled "Group 2"); and after partial elimination of metal carbonate particles from the particle-attached bacterial fraction by acid, i.e., from Group 2 (labeled "Group 3").

[0323] The results showed that culturing B. bifidum in the presence of the particle mixture resulted in increased attachment (at Figure 1 Group 2 cultured in the presence of 100% MCC particles produced an increase from Log 7.92 CFU / ml to Log 8.68-8.87 CFU / ml compared to cultured in the presence of both MCC and CaCO3).

[0324] The results also showed that ( Figure 1), culturing B. bifidum in the presence of the particle mixture resulted in an increase in total bacterial counts of -Log 9.35-9.49 CFU / ml (i.e., particle-attached and non-attached bacterial fractions; Group 1) compared to other bacterial growth forms: planktonic culture in the absence of particles (Log 8.10 CFU / ml) or culture in the presence of 100% MCC particles (Log 8.49 CFU / ml).

[0325] This result was confirmed by cultures of Lactobacillus crispatus and Lactobacillus paracasei, showing an increased trend in total bacterial counts and attachment levels in the combined particle-attached and non-attached bacterial fractions when cultured on the particle mixture (using the concentrations detailed above) compared to cultures on 100% MCC particles.

[0326] The results also showed that culturing various bacteria in the presence of 100% CaCO3 particles in the absence of MCC particles resulted in viscous media that often required increased stirring speeds to obtain a homogenous culture, negatively impacting the level of bacterial attachment to the particles.

[0327] It can also be seen that ( Figure 1 ), the addition of acid to the composition comprising the CaCO3-MCC particle mixture had no significant effect on the bacterial counts (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 even higher than the bacterial counts in the "attached fraction" of the MCC growth composition. The feasibility of using acid to eliminate CaCO3 particles while maintaining high bacterial counts was also demonstrated using cultures of Lactobacillus crispatus and Lactobacillus paracasei (data not shown).

[0328] For Lactobacillus crispatus, Lactobacillus paraceasi and Bifidobacterium bifidum, the pH value that obtains after adding the HCl of specified volume is 4.0.For Lactobacillus crispatus and Lactobacillus paraceasi (by the CO in solution Release observation), tested the HCl of extra volume, until calcium carbonate dissolves fully, this reaches the pH value that is lower than 3.0. Such low pH value level has reduced the bacterial survival rate by 14-45%. Advantageously, in order to obtain best bacterial survival rate, acid should be added with the amount that keeps the survival rate of bacteria substantially.

[0329] Example 2

[0330] Increased bacterial attachment to MCC-CaCO3 particle mixtures

[0331] In the following experiments, the effect of the ratio between the amount of particles and the volume of the culture medium on the level of bacterial attachment was examined.

[0332] Cultivation was performed in the presence of a 50%-50% MCC-CaCO3 pellet mixture and at different ratios (up to 200 g of pellets per 1 L of culture medium) (cultivation was performed as described in detail above).

[0333] The results showed that a ratio between 60 g of particle mixture per 1 L of medium, wherein calcium carbonate constituted 50% (w / w) of the total weight of the particles, and 200 g of particle mixture per 1 L of medium enhanced bacterial attachment to the particles (data not shown).

[0334] A composition according to an embodiment of the present invention may be prepared by contacting, incubating or culturing a population of at least one bacterial strain in the presence of particles comprising: (i) a polysaccharide-based material, such as MCC; and (ii) a metal carbonate, such as CaCO3, wherein the total weight of the particles relative to the volume of the culture solution may be in the range of between 60 g / 1L and 250 g / 1L, and wherein the metal carbonate may constitute between 15% w / w and 85% w / w of the total weight of the particles used, provided or added in the method.

[0335] Therefore, in order to obtain increased attachment, the ratio between the metal carbonate particles and the culture liquid used in the contacting process may advantageously be in the range of between 9 g / 1 L and 215 g / 1 L, taking into account the advantageous metal carbonate content range of between 15% and 85% w / w of the total weight of the total particles.

[0336] In methods wherein the particles further comprise a metal phosphate (eg DCP), the metal phosphate and metal carbonate may together constitute at least 15% w / w of the total weight of the particles. All weight ratios are based on the dry weight of the particles.

[0337] In general, incubation with metal carbonate concentrations above 85% (w / w of the total weight of the particles) at the particle:solution ratios specified above resulted in viscous cultures requiring increased stirring speeds to obtain a homogenous culture, which negatively impacted bacterial attachment to the particles. Furthermore, the use of metal carbonate concentrations below 15% (w / w of the total weight of the particles) resulted in reduced bacterial attachment.

[0338] Compositions can be made in which the incubation is carried out in the presence of particles having a total weight relative to the volume of solution of less than 60 g / 1 L. Under such conditions, the metal carbonate concentration can be between 15% and 99% (w / w) of the total weight of the particles used in the process.

[0339] Compositions prepared according to embodiments of the present invention may comprise attached and unattached bacterial components. Optionally, the two bacterial components may be separated, for example by filtration and / or gravity sedimentation, such as centrifugation as exemplified herein, and CFU measurements performed on each separated bacterial component.

[0340] To examine the effect of consumable calcium on bacterial attachment to particles, different bacterial populations were cultured in the presence of MCC particles in different media compositions with or without water-soluble calcium salts (CaCl2; 0.1%), and the effect of the presence of consumable calcium on the attachment level was examined.

[0341] The results indicated that including calcium ions in the culture medium did not enhance bacterial attachment to MCC particles (data not shown).

[0342] In summary, these results demonstrate the advantages of culturing in the presence of a mixture of particles according to the present invention compared to culturing in the presence of pure particles of MCC or CaCO 3 - achieving increased bacterial attachment to the particles and / or an increase in the total bacterial count of the composition. The results also demonstrate the feasibility of at least partially eliminating particles (e.g., CaCO 3 particles) from the bacterial composition during and / or after the culturing step while maintaining an increased bacterial count.

[0343] Example 3

[0344] Resistance of different bacterial compositions to external stress

[0345] The following examples examine the advantages of compositions prepared according to embodiments of the present invention in withstanding stress conditions compared to compositions of planktonic bacteria and / or bacterial compositions containing attached bacteria (96 w / p culture). The bacterial compositions were tested in both wet and lyophilized forms. Oxidative stress, a well-established assay, was selected as an exemplary stress condition.

[0346] Figure 2 Shown are the survival rates of wet (at the end of incubation) Lactobacillus plantarum cultures after exposure to increasing H2O2 concentrations. The resistance of attached bacterial compositions prepared by different incubation methods was tested: (i) incubation according to embodiments of the present invention in the presence of a CaCO3:MCC granular mixture (1:1 w / w ratio); and (ii) bacteria cultured on 96 w / p. Planktonic growth forms served as a reference. After H2O2 exposure, total bacterial counts (CFU) were measured. Results are expressed as a percentage compared to the respective control group ("no H2O2 addition" was considered 100%).

[0347] Figure 3 Shown are the survival rates of freeze-dried Lactobacillus plantarum cultures after reconstitution and exposure to increasing H2O2 concentrations. Resistance was tested in planktonic growth and culture in the presence of a CaCO3:MCC granular mixture (1:1 w / w ratio) according to embodiments of the present invention. Following H2O2 stress, total bacterial counts (CFU) were measured. Results are expressed as a percentage compared to the respective control group ("no H2O2 addition" was considered 100%).

[0348] The results show that cultivation in the presence of the particle mixture according to the invention has clear advantages in withstanding stress conditions compared to other cultivation methods, such as the production of compositions comprising an attached bacterial component, and compared to planktonic growth forms.

[0349] Although the present invention has been described in conjunction with the specific embodiments thereof, it is apparent that many alternatives, modifications and variations will be apparent to those skilled in the art. Therefore, the present invention is intended to cover 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-based material; and (ii) a metal carbonate.

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

3. The composition of claim 1 or 2, wherein the metal carbonate is not produced by the at least one bacterial strain.

4. The composition according to any one of claims 1 to 3, having at least one feature selected from the following: (a) comprising the particles in the form of a composite, the composite comprising the polysaccharide-based material and the metal carbonate; and (b) comprising each of the polysaccharide-based material and the metal carbonate in different particles.

5. The composition of any one of claims 1 to 4, wherein the particles further comprise dicalcium phosphate (DCP).

6. The composition according to any one of claims 1 to 5, further comprising particles free of polysaccharide-based materials and metal carbonates.

7. The composition according to any one of claims 1 to 6, wherein 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.

8. A composition according to any one of claims 1 to 7, wherein the polysaccharide-based material comprises a cellulose derivative.

9. The composition according to any one of claims 1 to 8, wherein the polysaccharide-based material comprises microcrystalline cellulose (MCC).

10. The composition of any one of claims 1 to 9, wherein the metal carbonate comprises calcium carbonate.

11. A composition according to any one of claims 1 to 10, comprising the metal carbonate in a concentration of up to 85% (w / w) 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 in that Any of the following: (i) comprising 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 comprising particles comprising a metal phosphate, and wherein 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 any one of claims 1 to 12, further comprising planktonic bacteria.

14. A composition according to any one of claims 1 to 13, comprising two or more bacterial strains.

15. A composition according to any one of claims 1 to 14 provided in solid form.

16. The composition according to any one of claims 1 to 15, further comprising a pharmaceutically acceptable carrier or excipient.

17. The composition according to any one of claims 1 to 16, for use in treating or preventing dysbiosis in a subject in need thereof.

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-based material; and (ii) a metal carbonate, the method comprising the steps of: providing a population of at least one bacterial strain; Providing a particle 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 attach to the particles; Thereby said composition is prepared, said composition comprising the population of said at least one bacterial strain at least partially attached to said particles.

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

20. The method of claim 18 or 19, wherein the contacting step comprises culturing a population of the at least one bacterial strain in a growth medium comprising the particles.

21. The method of any one of claims 18 to 20, wherein during the contacting step, the particles have at least one characteristic selected from the group consisting of: (a) being in the form of a complex comprising the polysaccharide-based material and the metal carbonate; and (b) comprising each of the polysaccharide-based material and the metal carbonate within different particles.

22. The method of any one of claims 18 to 21, wherein the provided particles further comprise dicalcium phosphate (DCP).

23. The method of any one of claims 18 to 22, wherein the weight of the metal carbonate relative to the volume of the solution is in the range of between 9 g / 1 L and 215 g / 1 L based on the dry weight of the particles.

24. The method according to any one of claims 18 to 22, wherein the total weight of the provided particles relative to the volume of the solution is in the range of between 60 g / 1 L and 250 g / 1 L based on the dry weight of the particles.

25. The method of claim 24, wherein the provided particles comprise the metal carbonate in a concentration of up to 85% (w / w) of the total weight of the particles based on the dry weight of the particles.

26. A method according to claim 24 or 25, wherein the provided particles are characterized by either: (i) comprising 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 comprising a metal phosphate, wherein 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 of any one of claims 18 to 26, wherein the metal carbonate is not produced by the at least one bacterial strain.

28. The method of any one of claims 18 to 27, further comprising the step of eliminating the metal carbonate from the particles by adding acid, CO2, or both.

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