Microbial compositions derived from fermented foods

Abstract

A method of generating a microbial composition is disclosed. The method comprises: (a) culturing at least one food source under conditions that promote native microbial growth; (b) separating resulting microbes from the cultured food to obtain a composition comprising at least 80% of the distinct microbial species present in the microbiota of the cultured food; and (c) processing the microbes by formulating the composition for oral, endoscopic, colonoscopic or rectal administration. Microbial compositions generated thereby and uses thereof are also disclosed.

Description

[0001] MICROBIAL COMPOSITIONS DERIVED FROM FERMENTED FOODS

[0002] RELATED APPLICATION / S

[0003] This application claims the benefit of priority of U. S. Provisional Patent Application No. 63 / 717,317 filed on November 7, 2024, the contents of which are incorporated herein by reference in their entirety.

[0004] FIELD AND BACKGROUND OF THE INVENTION

[0005] The present invention, in some embodiments thereof, relates to microbial compositions derived from food sources, and more particularly fermented foods.

[0006] The human gastrointestinal tract is host to a dense, diverse and dynamic ecosystem of microorganisms, collectively referred to as the gut microbiome. This community includes bacteria, archaea, viruses and fungi, which have evolutionary co-evolved with humans in a highly integrated symbiotic relationship. Among other essential physiological functions, the gut microbiome plays a critical role in digestion, immune modulation and maintenance of the intestinal barrier.

[0007] Recent advances in metagenomic sequencing have significantly expanded the known diversity of the human gut microbiota. The October 2025 release of the Unified Human Gastrointestinal Genome catalogue (UHGG v2.0) comprises over 289,000 prokaryotic genomes, grouped into 4,744 species-level clusters, isolated from stool samples across diverse geographic populations. In a typical adult, the gut harbors approximately 200–300 bacterial species, representing a microbial biomass of roughly 1–2 kilograms. While bacteria dominate in both richness and mass, the gut microbiome also includes lesser quantities of archaeal species, fungal taxa, and various viruses.

[0008] A diverse and well-balanced gut microbiome is widely associated with positive health outcomes. It confers “colonization resistance,” a phenomenon wherein resident commensals competitively exclude pathogenic microbes. Moreover, the microbiota interacts closely with the host’s immune system, reinforcing gut epithelial integrity and regulating inflammatory responses. Microbial metabolites, enzymes, and fermentation intermediates produced by the gut microbiota are considered integral components of the host's metabolic landscape, collectively referred to as the gut metabolome.

[0009] However, disturbances to this microbial equilibrium - termed “dysbiosis” - are increasingly recognized as contributing factors in a wide array of diseases. Dysbiosis involves measurable shifts in microbiome structure, typically characterized by reductions in taxonomic richness and evenness (a-diversity), or altered compositional similarity across individuals or populations (β-diversity). Dysbiosis has been implicated in gastrointestinal disorders such as Irritable Bowel Syndrome (IBS), Inflammatory Bowel Disease (IBD), and antibiotic-associated overgrowth of multidrug-resistant organisms (MDROs). Beyond the gut, dysbiosis has also been associated with neurodegenerative diseases (e.g., Parkinson’s disease, Multiple Sclerosis), obesity, metabolic syndrome, immune dysregulation, and systemic inflammation.

[0010] A major iatrogenic driver of dysbiosis is antibiotic exposure, which indiscriminately eradicates both pathogenic and beneficial bacterial taxa. One particularly concerning outcome is Clostridioides difficile infection (CDI), which often arises in community and hospitalized patients following antibiotic therapy. C. difficile can persist in the gut through the formation of resilient endospores, leading to recurrent infections (rCDI) that are difficult to treat and can be fatal.

[0011] Fecal Microbiota Transplantation (FMT) has emerged as a leading bacteriotherapy for rCDI and other forms of dysbiosis. While effective in many cases, FMT is currently classified as an Investigational New Drug (IND) by the U. S. Food and Drug Administration (FDA), and is subject to strict regulatory oversight. Importantly, FMT carries inherent risks, including the potential for transmission of infectious agents from donor to recipient, even under rigorous donor screening protocols. In response to adverse events, the FDA has issued multiple safety warnings regarding FMT-related pathogen transmission.

[0012] In addition to safety concerns, many patients find FMT aesthetically and psychologically unappealing. Delivery methods are typically invasive and may include colonoscopy, transnasal endoscopy, sigmoidoscopy, or rectal enema. Pharmaceutical efforts to develop rationally designed microbial consortia, or defined synthetic formulations, have made some progress, but most remain derived from stool donors and may still rely on invasive delivery.

[0013] One oral alternative is the FDA-approved spore-based human stool-derived product Vowst™ (Ser- 109), indicated for rCDI. However, Vowst™ is limited in both microbial diversity and accessibility. Its microbial composition includes only ~50 bacterial spore-forming strains, all from the single phylum Firmicutes, and the cost of a single treatment course is approximately $17,500 USD. Another FDA-approved human stool product is Rebyota™ (RBX2660), an undisclosed composition requiring rectal administration, the cost of which amounts to 9,500 USD.

[0014] SUMMARY OF THE INVENTION

[0015] According to an aspect of some embodiments of the present invention there is provided a method of generating a microbial composition comprising:

[0016] (a) culturing at least one food source under conditions that promote native microbial growth; (b) separating resulting microbes from the cultured food to obtain a composition comprising at least 80% of the distinct microbial species present in the microbiota of the cultured food; and (c) processing the microbes by formulating the composition for oral, endoscopic, colonoscopic or rectal administration.

[0017] According to embodiments of the invention, the microbes are formulated as a capsule, as a tablet, as a dry powder, as a suppository or as a suspension.

[0018] According to embodiments of the invention, the processing comprises freezing, drying or homogenization.

[0019] According to embodiments of the invention, the culturing is affected at a pH level that inhibits the growth of pathogenic microorganisms.

[0020] According to embodiments of the invention, the separation step comprises filtering, centrifugation, or decantation.

[0021] According to embodiments of the invention, the at least one food source is a lactic acid fermentable food substrate.

[0022] According to embodiments of the invention, the cultured food is a plant-based food, algae, an animal-based food or kefir grains.

[0023] According to an aspect of some embodiments of the present invention there is provided a composition comprising microbes, wherein the composition is generated according to the methods described herein.

[0024] According to embodiments of the invention, the microbes are viable.

[0025] According to embodiments of the invention, at least 80 % of the resulting microbes in the composition originate from the fermented food source.

[0026] According to embodiments of the invention, the composition is non-caloric.

[0027] According to embodiments of the invention, the relative abundance distribution of the microbial species in the composition is substantially similar to the distribution of the microbial species in the microbiota of the food source following the culturing.

[0028] According to an aspect of some embodiments of the present invention there is provided a non-caloric composition comprising a microbial population that comprises at least 80% of the distinct microbial species that are present in the microbiota of at least one fermented food; the composition being formulated for oral, endoscopic, colonoscopic or rectal administration.

[0029] According to embodiments of the invention, at least 50 % of the microbes of the non-caloric composition originate from the food source.

[0030] According to an aspect of some embodiments of the present invention there is provided a non-caloric composition comprising a microbial population that comprises at least 10 distinct microbial species present in the microbiota of a fermented food, the composition being formulated for oral, endoscopic, colonoscopic or rectal administration. According to embodiments of the invention, the 10 distinct microbial species originate in the food source of the fermented food.

[0031] According to embodiments of the invention, least 50 % of the microbes of the non-caloric composition originate from the food source.

[0032] According to embodiments of the invention, the composition is formulated as a capsule, a tablet, dry powder, a suspension or a suppository.

[0033] According to embodiments of the invention, the relative abundance distribution of the microbial genera in the microbial population of the fermented composition is substantially similar to the distribution of the microbial genera in the microbiota of the food source following culturing.

[0034] According to embodiments of the invention, the food source is a natural food source.

[0035] According to embodiments of the invention, the food source is a lactic acid fermentable food substrate.

[0036] According to embodiments of the invention, the food source is a plant.

[0037] According to embodiments of the invention, the food source comprises a fruit or a vegetable. According to embodiments of the invention, the vegetable comprises a cruciferous vegetable. According to embodiments of the invention, the food source comprises Kefir grain.

[0038] According to embodiments of the invention, the microbial population comprises bacteria. According to embodiments of the invention, the microbes of the microbial population are viable.

[0039] According to embodiments of the invention, the non-caloric composition comprises at least 10 different species of bacteria present in the microbiota of the same fermented food.

[0040] According to embodiments of the invention, the non-caloric composition comprises at least 20 different species of bacteria present in the microbiota of the same fermented food.

[0041] According to an aspect of some embodiments of the present invention there is provided a non-dairy composition comprising a microbial population that comprises at least 80% of the distinct microbial species that are naturally present in the microbiota of Kefir grains, wherein at least 50 % of the microbes of the non-dairy composition originate from the Kefir grains when cultured in milk, the composition being formulated for oral, endoscopic, colonoscopic or rectal administration.

[0042] According to embodiments of the invention, the composition is formulated as a capsule, a tablet, a dry powder, a suspension or a suppository.

[0043] According to embodiments of the invention, the composition is homogenized, dried or frozen. According to embodiments of the invention, the relative abundance distribution of the microbial species in the microbial population is substantially similar to the distribution of the microbial species in the microbiota of the Kefir grains following fermentation.

[0044] According to embodiments of the invention, the non-dairy composition comprises kefir grains washed away from milk.

[0045] According to embodiments of the invention, the microbes of the microbial population are viable.

[0046] According to embodiments of the invention, the non-dairy composition comprises at least 5 distinct species of yeast.

[0047] According to embodiments of the invention, the non-dairy composition comprises at least 20 distinct species of bacteria.

[0048] According to embodiments of the invention, the composition is devoid of fecal material. According to embodiments of the invention, the composition is devoid of pathogenic taxa. According to another aspect of the invention, there is provided a fortified food comprising the composition described herein.

[0049] According to embodiments of the invention, the composition is for use in treating a disease associated with dysbiosis.

[0050] According to an aspect of some embodiments of the present invention there is provided a method of treating a subject having a disease associated with dysbiosis of the gut microbiome comprising administering to the subject a therapeutically effective amount of the composition or fortified food described herein, thereby treating the subject.

[0051] According to embodiments of the invention, the disease is a metabolic disorder.

[0052] According to embodiments of the invention, the metabolic disorder is selected from the group consisting of obesity, metabolic syndrome, type 2 diabetes (NIDDM) and metabolic associated fatty liver disease (MAFLD).

[0053] According to embodiments of the invention, the disease is a neurological or psychiatric disorder.

[0054] According to embodiments of the invention, the disease is a gastrointestinal disorder.

[0055] According to embodiments of the invention, the gastrointestinal disorder is selected from the group consisting of inflammatory bowel disease (IBD), irritable bowel syndrome (IBS) and colorectal cancer (CRC).

[0056] According to embodiments of the invention, the disease is an autoimmune and inflammatory disease. According to embodiments of the invention, the autoimmune and inflammatory disease is selected from the group consisting of rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), celiac disease and psoriasis.

[0057] According to an aspect of some embodiments of the present invention there is provided a method for restoring gut microbial diversity in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition described herein following antibiotic treatment.

[0058] According to embodiments of the invention, the composition is for use in restoring gut microbial diversity following antibiotic treatment.

[0059] According to embodiments of the invention, the composition is administered by the oral, endoscopic, colonoscopic or rectal route.

[0060] According to embodiments of the invention, the composition is administered within 1 to 14 days following completion of antibiotic therapy.

[0061] According to embodiments of the invention, the administration of the composition reduces gastrointestinal side effects associated with antibiotic therapy.

[0062] According to an aspect of some embodiments of the present invention there is provided a method of maintaining the health of a subject comprising administering to the subject a therapeutically effective amount of the composition described herein, thereby maintaining the health of the subject.

[0063] According to embodiments of the invention, the administering comprises orally administering.

[0064] According to an aspect of some embodiments of the present invention there is provided a method of treating a subject having a disease for which immunotherapy is therapeutically beneficial, comprising administering to the subject a therapeutically effective amount of the immunotherapy and the composition described herein, thereby treating the subject.

[0065] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0066] The present invention, in some embodiments thereof, relates to microbial compositions derived from food sources in general and more specifically from fermented foods.

[0067] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0068] The present inventors have conceived of a therapeutic solution to gut dysbiosis, which entails reconstituting a healthy gut microbiome from safe, non-human sources. The invention applies to several therapeutic domains, such as providing a diverse microbial composition for daily maintenance of gut health and treatment of gut pathologies. The present inventors proposed that the disclosed microbial compositions can support transient and competitive colonization of vacant niches in the gut, thus enabling the gradual rebound of healthy human commensal microbiome, over time.

[0069] While microbiota taxa generated during native fermentation of food sources is not identical to taxa found in the human GI tract, several mutual genera and functional overlaps exist. Fermented foods have been shown to transiently colonize the human gut, thus paving way to their use for competitive colonization of depleted gut niches. Following such transient colonization, gradual commensal long-term colonization can take place.

[0070] The benefit of a microbial consortia derived from fermentation of food sources, can be reductively assessed relying on relative abundance of its phyla, genera, species or strains. However, this approach falls short of covering the interrelations and cross-feeding mechanisms which exist in complex consortia. In fact, the transcriptome and metabolome of a consortia plays a pivotal role in sustaining and preserving the attributes of that consortia. Contrary to isolated, rationally designed strains, which are cultured in synthetic media devoid of any selective pressures, the native consortia maintains its metabolic integrity due to an ongoing metabolite crosstalk between members of the community. Down regulation, loss of function and genomic instability, have also been reported in isolated strain amplification.

[0071] Spontaneous and native lactic acid fermentation entails both homofermenation to yield lactic acid (the ultimate preserver) and heterofermentation to yield a plethora of metabolites required for enteroctyte health and crosstalk between the growing microbial community strains. Contrary to the human gut, food ferments may support oxygen-tolerant and facultative bacteria, which are more amenable to isolation and purification under non-stringent conditions. Several food ferment derived strains are also acid tolerant, making them excellent candidates for oral administration. Quite differently from direct consumption of crude fermented foods, the isolation and purification of microbiota from fermented foods, offers a clean, diverse and almost calorie-free consortia of diverse microorganisms, purified away from potential food-borne antinutrients and allergens. Some members listed under this group of aggravators are histamines, tyramines, lectins, phytates, oxalates, tannins, saponins, protease inhibitors, goitrogens, isoflavones, lactose and fiber. All of the listed pose a profound dietary obstacle, exactly to those afflicted with pathological dysbiosis and most needy of a diverse microbiome.

[0072] More specifically, the invention relies on extracting microbiota from fermented foods, as fermented foods portray amplification and expansion of naturally inherent taxa of native microorganisms carried on the raw food sources. By combining microbiota sourced from a number of fermented foods, the amount and diversity of microorganisms can be increased and widened.

[0073] Whilst conceiving embodiments of the present invention, the present inventors have analyzed the microbial content of two exemplary fermented foods - Kefir grains and Sauerkraut. As summarized in Table 1, both of these foods contain a wide diversity of taxa.

[0074] The present inventors further propose inclusion of Kefir grains themselves in the composition. The grains comprise a Kefiran polysaccharide scaffold which serves as a prebiotic for the microbes therein, classifying it as a symbiotic composition.

[0075] Thus, according to an aspect of the invention, provided is a method of generating a microbial composition comprising:

[0076] (a) culturing / fermenting at least one food source under conditions that promote native microbial growth;

[0077] (b) separating the resulting microbes from the cultured food, to obtain a composition comprising at least 80% of the distinct microbial species present in the microbiota of the cultured food; and

[0078] (c) processing the separated microbes by formulating the microbial composition for oral, endoscopic, colonoscopic or rectal administration.

[0079] The term, “microbial composition”, as used throughout the specification, refers to a collection of microbial entities, including viable cells, spores, phages, or structural fragments thereof-comprising one or more microbial taxa, typically at the genus, species, or strain level. The composition may consist of a single microbial type or a complex community of multiple microbial genera or species contributed by one or a number of food ferments, and may include bacteria, archaea, fungi, molds, viruses, phages, or other microscopic organisms. In one embodiment, the composition comprises microbes which are derived from the native fermentation processes, without any externally assisted inoculation. In some embodiments, the microbial composition is characterized by its taxonomic structure, including the relative abundance or presence of specific genera or species, or by its functional attributes, such as metabolite production, probiotic activity, bile acid conversion, resistance to gastric acidity, resistance to antibiotics, or colonization capability. The composition may be naturally derived, synthetically assembled, or enriched from a biological source, and may include both cultivable and uncultivable microbes as determined by culture-independent techniques (e.g., 16S rRNA sequencing or metagenomic analysis).

[0080] In some embodiments, rationally designed and in vitro cultured strains may be added to the consortia of microorganisms which were derived and purified from the fermented food.

[0081] The term “native microbial growth” refers to microbes that are native to the food itself and are not derived from the processing environment (i.e., those that inhabit the surface, interior, or immediate environment of the food source).

[0082] All the microbial compositions described herein are distinct from fecal transplants in both origin and composition. Unlike fecal transplants, which are derived from the heterogeneous and donor- specific microbiota of human stool, the present compositions are generated from non-fecal food sources and contain microbial genera that are naturally present in the microbiota of those foods.

[0083] As used herein, the term “food source” refers to any material or substance that can be ingested by an organism (e.g., human) to provide nutritional support, including but not limited to carbohydrates, proteins, fats, vitamins, minerals, and water. The food source may be derived from plant, algae, animal, microbial or synthetic origin, and may include whole foods and processed food products. In some embodiments, the food source may be solid, semi- solid or liquid. The food source refers to the food prior to any culturing / fermenting step.

[0084] In one embodiment, the food source is washed so as to remove contaminating microbes on the surface thereof.

[0085] In another embodiment, the food source is stored under conditions that minimize microbial contamination.

[0086] In other embodiments, the food source is grown under controlled conditions, such as clean rooms.

[0087] The microbial composition described herein may be derived from a single fermented food or from a combination of multiple fermented foods, each of which contributes a distinct microbiota. In some embodiments, the composition is obtained by culturing / fermenting a single, food material (e.g., a fruit, vegetable, grain, meat, fish or dairy product) under conditions that promote the proliferation of its naturally present microbial community. In such cases, the resulting microbial composition reflects the ecological and taxonomic structure inherent to that single food substrate.

[0088] In other embodiments, the microbial composition is derived from two or more distinct fermented foods, which may be selected to provide complementary microbial profiles or functional diversity. When multiple food sources are used, the method is carried out such that at least 80%, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or even 100 % of the distinct microbial species present in the microbiota of each fermented foods are preserved in the final composition. This ensures that the contribution of each fermented food-derived microbiome is retained in a representative and taxonomically diverse manner, avoiding dominance by one source at the expense of others.

[0089] Exemplary contemplated combinations include, but are not limited to Sauerkraut and Kefir grains. Typical of these sources is their inherent content of microorganisms, giving rise to native fermentation, without external inoculum. This exemplary combination is also a symbiotic combination, brought by the kefiran prebiotic. For live microbiota extraction, all fermented foods are typically devoid of end-product pasteurization or end-product inactivation. Thus, additional plausible combination partners, arising from native / spontaneous fermentation, may include: Kimchee, Paocai, Nozau-zuke, Taar, Kali, Boza, Khalpi, Inziangsang, Soidon, Goyang, Miso, Sougherdough, Idli, Ogi, Pickeled Vegetables, GInjera, Kvass, Traditional Fish Pastes, Fermented Meat and Game Meat, Kiviak, Menju, Doenjang, Makgeolli, Pulque, Tepache, Chicha, Cauim, Masato, Gari, Boza, Rejuvelac, Tempeh, Natto, Kinema, Borsh, Way-a-Linah, Mangaitch, Kambuda, Tuba, Trahanas, Kefir, Kefir SCOBY, Water Kefir, Water Kefir SCOBY, Kombucha, Kombucha SCOBY, Qurut, Shubat, Certain Artisan Cheese, Butter and Cream ferments.

[0090] Each food source may be cultured / fermented separately, allowing for precise control over growth conditions tailored to the microbial ecology of that source. In such embodiments, the separately cultured microbial populations may be combined after separation to form a blended composition. Alternatively, the food sources may be co-cultured / fermented, wherein the raw food materials are combined prior to, or during incubation under shared conditions. Co-culturing may facilitate natural microbial interactions across substrates and may lead to emergent microbial profiles that reflect interspecies interactions across food sources.

[0091] Regardless of whether separate or co-culturing is used, the method is implemented such that the microbial species native to each fermented food source are not substantially depleted or excluded during the separating step, or during formulation. The preservation of at least 80% of the species from each fermented food in the composition, helps to maintain the functional and ecological integrity of the composition and supports the goal of delivering a broad, diverse, and reproducible microbiota for therapeutic or nutritional applications.

[0092] According to a particular embodiment, the food source is not of synthetic origin (i.e., is a natural food source).

[0093] In one embodiment, at least one food source is a lactic acid fermentable food substrate. The phrase “lactic acid fermentable food substrate” refers to any food material that includes fermentable carbohydrates and is suitable for microbial fermentation supporting both homofermentation to yield lactic acid and heterofermentation to yield Short Chain Fatty Acids (SCFA) and other beneficial metabolites. Suitable substrates may include raw, partially cooked, cooked, or minimally processed plant, algae or animal-derived foods that support lactic acid bacterial growth and metabolic activity.

[0094] In some embodiments, plant-based substrates may include:

[0095] Vegetables, or closely related to plants, such as cruciferous vegetables (e.g., cabbage), carrots, cucumbers, beets, radishes, leafy greens or algae;

[0096] Fruits, such as apples, berries, pineapples, coconut flesh, tomatoes and bananas;

[0097] Cereal grains and legumes, such as rice, wheat, oats, com, millet, soybeans and chickpeas; Plant juices or purees, including sugar-rich extracts like sugarcane or fruit juices.

[0098] In other embodiments, animal-based substrates may include:

[0099] Milk, dairy products and their analogs, including cow, goat, or non-pasteurized plant-based milks;

[0100] Meat or fish, especially when combined with carbohydrate-containing additives to promote fermentation (e.g., in traditional fermented sausages or fish sauces).

[0101] In certain embodiments, the food substrate may further include added fermentable sugars, such as glucose, fructose, or sucrose, to optimize fermentation efficiency and the speed of acidification.

[0102] In some embodiments, the lactic acid fermentable food substrate may be co-fermented with kefir grains, which is a SCOBY comprised of a symbiotic microbial consortium of lactic acid bacteria, yeasts, and acetic acid bacteria embedded in a polysaccharide matrix. In such embodiments, kefir grains may act both as a native fermentation starter and as a modulator of substrate properties, enabling the fermentation of milk, water-sugar solutions (e.g., water kefir), or other suitable carbohydrate-containing media.

[0103] In all cases, the substrate should contain, or be supplemented with, sufficient levels of available simple or complex carbohydrates (e.g., at least 0.5%, at least 1%, at least 2%, at least 5%, or at least 10% w / w) to enable measurable lactic acid production during fermentation. Foods can be cultured in any way so as to expand the naturally occurring population of selected microbes within. The duration of culture may be between 1 hour to 1 week, or more, depending on the substrate type.

[0104] In one embodiment, the culturing comprises fermenting.

[0105] The term “fermenting” as used herein refers to a process in which microbial activity is utilized to biochemically transform a food substrate, typically through the metabolic conversion of carbohydrates, proteins, or other organic compounds into products such as acids, gases, alcohols, or flavor compounds. Fermentation involves the proliferation of specific microbial populations - often lactic acid bacteria, yeasts, fungi, or molds, under conditions favorable to their growth, including appropriate temperature, moisture, pH, and, in many cases, anaerobic or semi-anaerobic environments.

[0106] In one embodiment, fermentation does not involve further in vitro culturing of the end-point food ferment, as this may bring about changes in the communal composition of the consortia with potential changes in genetic composition and metabolome profile.

[0107] In one embodiment, the fermentation relies on microbes already present in the food.

[0108] In another embodiment, the end product of a spontaneous fermentation product is fortified with rationally designed bacterial taxa which are grown separately and synthetically.

[0109] In some embodiments, the richness and evenness of a certain ferment may be defined statistically by its Shannon Diversity Index, whereas similarity or dissimilarity between 2 different ferments may be defined statistically, for example, by Bray-Curtis dissimilarity index, Jaccard similarity index, or another ecological metric.

[0110] In a specific embodiment, the at least one food source is cultured so as to expand microbes which are capable of producing lactic acid accompanied by a concomitant pH drop, thus suppressing undesirable microbial growth. This typically involves creating favorable ecological conditions for lactic acid bacteria (LAB), including adequate carbohydrate availability, moderate temperatures (e.g., 18-35°C), and, where appropriate, anaerobic or microaerophilic environments. In many traditional fermentations, natural microflora already present on the food substrate or in the environment initiates the process, as seen in the fermentation of vegetables (e.g., sauerkraut, kimchee), cereals (e.g., ogi, idli), or cassava (e.g., gari). The use of salt, as in the brining of cabbage or cucumbers, serves both to extract water and inhibit spoilage organisms. Temperature and salt concentration critically influence the succession and dominance of microbial species. For instance, Leuconostoc mesenteroides initiates vegetable fermentations under lower salt and temperature conditions, followed by acid-tolerant species like Lb. plantarum. Select fermentations may also incorporate natural inocula, such as kefir grains or spontaneously fermented mash, to standardize, diversify and expedite microbial succession.

[0111] The duration of lactic acid fermentation varies by substrate type, sugar content, initial microbial load, temperature and salt concentration. For example, sauerkraut fermentation may take 8 to 20 days at 18-32°C, whereas kimchee can reach optimal acidity in 3 to 7 days at ~10°C. Pickled cucumbers may ferment over two weeks. Cereal-based fermentations like idli or ogi typically require 12 to 48 hours, depending on temperature and grind. In cassava fermentations such as gari, a 12-96 hour period is typical. Milk fermented with kefir grains is usually ready in 24 to 48 hours, while pulque may take 1 to 3 days. The endpoint of fermentation is typically indicated by reaching a target pH (e.g., 3.5 to 4.5), desired titratable acidity, or characteristic organoleptic properties.

[0112] Exemplary conditions for lactic acid fermentation are further described in Applications of Biotechnology in Traditional Fermented Foods, National Academy press, Washington DC, 1992, the contents of which are incorporated herein by reference.

[0113] In another embodiment, the conditions under which the at least one food source is cultured, ensure that at least 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more genera of bacteria that were present in the original food source remain viable in the culture.

[0114] In still another embodiment, the conditions under which the at least one food source is cultured ensure that at least 10, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 50 or more species of bacteria that were present in the original food source remain viable in the culture.

[0115] In still further embodiments, the conditions under which the at least one food is cultured ensure that at least 5, 10, 15 or 20 or more species of yeast that were originally present in the food source remain viable in the culture.

[0116] Separating the resulting microbes from the cultured food material to obtain a composition comprising at least 80 % of the distinct microbial species present in the microbiota of the cultured / fermented food may be affected by physical, mechanical, or biochemical means. In some embodiments, the separation includes mechanical disintegration of the cultured substrate followed by crude prefiltration, filtration, centrifugation, decantation, or density gradient separation to isolate microbial cells from the food matter.

[0117] In a particular embodiment, prefiltration of the crude material may be performed by using devices equipped with a sterile straining mesh, muslin, gauze or sterile cloth. In a particular embodiment, the cultured food is placed into a stomacher-type sterile filter bag fitted with a porous membrane of approximately 63 microns, and mechanically agitated in a sterile buffered solution using a paddle blender or stomacher device. The filter allows microbial cells and small aggregates to pass through, while retaining larger food particulates, thereby producing a clarified microbial suspension that preserves microbial diversity. Such a suspension can be further concentrated by centrifugation or density gradient separation.

[0118] In alternative embodiments, equivalent separation devices may be used, such as nylon mesh bags, filter-lined centrifuge tubes, or reusable filtration cartridges, having pore sizes ranging, for example, from 25 to 100 microns, or more preferably 40 to 80 microns, depending on the physical characteristics of the food matrix and target organisms. These methods are selected to also allow enrichment of aggregates or chains of bacteria (e.g., streptococcus) thus avoiding selective enrichment, preserving both robust and fastidious genera.

[0119] In other embodiments, separation may involve washing steps using sterile buffered solutions to detach surface-adhered microbes, followed by differential sedimentation or membrane-based fractionation.

[0120] In some embodiments, the separation is carried out using non-selective recovery techniques designed to minimize taxonomic bias. In some embodiments, the cultured food is homogenized mechanically, chemically, or enzymatically to release microbial cells from the food source without significantly disrupting microbial viability or selectively enriching certain taxa.

[0121] The term “non-selective recovery” as used herein refers to processing methods that do not favor specific genera or phyla, thereby preserving the natural relative abundance and diversity of the microbiota in the fermented product. To minimize taxonomic bias, the separation step may avoid harsh conditions (e.g., high heat, oxidative exposure, selective culture media) that disproportionately affect fastidious, anaerobic, or less robust genera. In some embodiments, oxygen-sensitive microbes are recovered using anoxic buffer solutions or conducted in anaerobic chambers.

[0122] Typically, at least 50 %, 60 %, 70 %, 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or even 100 % of the microbial genera / species present in the final composition originate (i.e., are present or are progeny of those that are present) from the food source which is being cultured. In some embodiments, the separation is performed in a manner that preserves anaerobic or facultative anaerobic organisms by minimizing oxygen exposure. The recovered microbial composition may be stored or directly processed for downstream formulation. In a particular embodiment, the separation is carried out such that the resulting microbial composition is substantially depleted of digestible or metabolizable caloric substrates, and may therefore be defined as virtually non-caloric.

[0123] In a particular embodiment the separation is carried out such that the resulting microbial composition is substantially depleted of allergens and antinutrients.

[0124] As used herein, the term “non-caloric” refers to a composition that contributes no more than 10 kilocalories (kcal) per typical human dose or serving, based on digestible macronutrients (carbohydrates, proteins, and fats) as defined by standard food energy calculation models. In some embodiments, the non-caloric status may be achieved by removing or excluding residual food particulates, soluble sugars, lipids, or peptides that are metabolizable by the human digestive system. In other embodiments, caloric content may be minimized through washing, filtration, centrifugation, enzymatic degradation, or dialysis.

[0125] The resulting composition may consist predominantly of microbial biomass (e.g., intact cells or spores) and cell fragments, with minimal microbial metabolites (e.g., bacteriocins, SCFA, cell wall components), without co-delivery of caloric nutrients, allergens and antinutrients derived from the original food substrate.

[0126] Following the separation phase, the microbes are processed by formulating the composition for oral, endoscopic, colonoscopic or rectal administration.

[0127] As mentioned, in some embodiments, the microbial compositions described herein may include additional microbial strains or species that are not originally present in the food source microbiota. These may be added, for example, to enhance functionality, stability, or therapeutic efficacy of the final formulation. However, in all such embodiments, the composition remains predominantly food-derived. This ensures that the added organisms do not substantially alter the native ecological community structure or overwhelm the taxonomic character of the composition as derived from the fermented food substrate.

[0128] Quantitative assessment of the microbial population may be performed by total cell counts, live cell counts while using a viability exclusion dye, relative abundance from sequencing data, or colony-forming unit (CFU) enumeration of representative taxa lending themselves to in vitro culturing, depending on the context.

[0129] Processing the microbes by formulating the composition for oral, endoscopic, colonoscopic or rectal administration may be carried out using methods designed to preserve and deliver viable microorganisms to the gastrointestinal tract. In some embodiments, the separated microbial composition is concentrated, washed, and stabilized under conditions that maintain cell integrity and viability, followed by formulation into an ingestible form. Suitable dosage forms may include, without limitation: purees, capsules, acid-resistant enteric capsules, tablets, dry powders, suppositories, suspensions, emulsions, gels, lozenges, sachets, or oral sprays.

[0130] Processing may involve freezing the microbial composition, either as an intermediate stabilization step or as a long-term storage method. Freezing may be conducted using controlled-rate freezing or cryoprotectants to minimize ice crystal damage to microbial cells.

[0131] In some embodiments, the microbial composition is subjected to controlled homogenization to achieve uniform particle distribution and consistent microbial load across the formulation. Homogenization may be carried out using low-shear mixing, rotor-stator systems, or pneumatic or hydraulic blending devices, depending on the viscosity and volume of the microbial suspension. To preserve microbial viability, homogenization may be performed at refrigerated temperatures (e.g., 2-10 °C) and under anaerobic or low-oxygen conditions, particularly when the microbial community includes oxygen- sensitive genera. In one embodiment, a gentle rotor-stator homogenizer is used to create a uniform suspension of microbial cells in a buffered carrier fluid prior to encapsulation or drying. In another embodiment, paddle-type or planetary mixers are used to blend microbial biomass with protective excipients such as sugars, prebiotics, or encapsulation agents. The homogenization parameters - such as time, shear rate, and energy input - are selected to avoid cell lysis or membrane damage, thereby maintaining viability while ensuring compositional consistency. If needed, process validation may be performed using viability assays before and after homogenization to confirm preservation of cell integrity.

[0132] In one embodiment, the microbial composition is lyophilized (freeze-dried), for example in the presence of viability-preserving excipients, such as trehalose, sucrose, or skim milk powder, to protect microbial cells during drying and storage. The lyophilized material may be encapsulated in acid-resistant or enteric-coated capsules to shield the microbes from gastric acid and deliver them to the intestine. Alternatively, the composition may be formulated into anhydrous powder blends, for rehydration before use.

[0133] Processing is preferably carried out at low temperatures and in oxygen-reduced environments where necessary, particularly for preserving anaerobic or facultative anaerobic genera. In some embodiments, microencapsulation (e.g., in alginate, chitosan, or lipid vesicles) is employed to further protect microbial viability and facilitate targeted release.

[0134] Throughout the processing workflow, conditions may be optimized to minimize thermal, oxidative, osmotic, or mechanical stress that could reduce microbial viability. The resulting formulation includes a viable microbial population corresponding to the taxonomic profile of the original culture / ferment, optionally verified by viability assays such as colony-forming unit (CFU) counts of representative taxa, flow cytometry with viability stains, or respirometry. According to another aspect of the invention, there is provided a non-caloric composition comprising a microbial population that comprises at least 80 % of the distinct microbial species that are present in the microbiota of at least one fermented food;

[0135] wherein at least 50 % of the microbes of the microbial population originate from the native fermented food source, the composition being formulated for oral, endoscopic, colonoscopic or rectal administration.

[0136] As used herein, the phrase “naturally present in the microbiota of a fermented food” refers to microbial genera that are part of the indigenous, microbial community associated with the fermented food. This includes microorganisms that inhabit the surface, interior, or immediate environment of the food source.

[0137] In this context, a natural food source refers to any unprocessed or minimally handled biological material derived from plant, animal, fungal, algae, or microbial origin, including but not limited to fresh fruits, vegetables, grains, seeds, roots, tubers, milk, or wild-caught seafood, provided it is not subjected to prior fermentation, pasteurization, chemical treatment, or intentional microbial seeding. For example, the native microbial community present on the skin of a raw apple, the outer leaves of cabbage, or freshly harvested grains would qualify as naturally present. Other examples of foods sources are described herein above.

[0138] In this context, kefir grains by themselves can classify as a food source, requiring natural media (milk or water-based sugary solutions) for culturing / fermenting / back-slopping.

[0139] As mentioned, at least 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or even 100 % of the distinct microbial species that are naturally present in the microbiota of at least one fermented food are present in the composition, as described herein above.

[0140] Furthermore, at least 50 %, 60 %, 70 %, 60 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or even 100 % of the microbes of the composition originate from the fermented food source, as described herein above.

[0141] In another embodiment, the conditions under which the food is cultured ensure that at least 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more species of bacteria that were present in the food source remain viable in the culture.

[0142] In still another embodiment, the conditions under which the food is cultured ensure that at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60 or more species of bacteria that were present in the food source remain viable in the culture.

[0143] Exemplary bacterial and yeast species which may be found in the composition made from a food ferment are summarized in the Example section, herein below. In still further embodiments, the conditions under which the food is cultured ensure that at least 5, 6, 7, 8, 9, 10 or more genera of yeast that were originally present in the food source remain viable in the culture.

[0144] In still further embodiments, the conditions under which the food is cultured ensure that at least 5, 10, 15 or 20 or more species of yeast that were originally present in the food source remain viable in the culture.

[0145] According to still another aspect, there is provided a non-caloric composition comprising a microbial population that comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, or over, distinct microbial species present in the microbiota of the food ferment (typically originating from the food source), being formulated for oral, endoscopic, colonoscopic or rectal administration. Typically, at least 50 %, 60 %, 70 %, 80 %, 90 % or higher % of the microbes of this composition originate in the fermented food.

[0146] For any of the compositions described herein, exemplary food sources are provided above. Methods suitable for producing any of the compositions disclosed herein are described in preceding sections.

[0147] Representative formulations of any of the disclosed compositions are set forth above.

[0148] For any of the compositions described herein, typically, no more than 10, 15, 20, 25, 30, 35, 30, 45, 50, 60, 70, 80, 90 or 100 microbial species are present in the compositions.

[0149] For any of the compositions described herein, typically, no more than 10, 15, 20, 25, 30, 35, 30, 45, 50, 60, 70, 80, 90 or 100 microbial species are present in the compositions per food source. Exemplary ranges include 5-50, species, 5-100 species, 5-30 species.

[0150] In one embodiment, the food source of any of the disclosed compositions is cultured (e.g., fermented), as described herein above.

[0151] In some embodiment the composition is made from one, or more than one food source, or fortified with an in vitro cultured strain / strains.

[0152] According to still another aspect of the invention there is provided a non-dairy composition comprising a microbial population that comprises at least 80% of the distinct microbial species that are naturally present in the microbiota of Kefir grains, wherein at least 50 % of the microbes of the composition originate from the Kefir grains when cultured in milk, the composition being formulated for oral, endoscopic, colonoscopic or rectal administration. The non-dairy assignment of kefir-grain-derived microbiota stems from the ability to wash away milk from the grains.

[0153] As used herein, the term “non-dairy composition” refers to any ingestible or administrable formulation that does not comprise mammalian milk or a mammalian milk product (e.g., yoghurt or cheese). In some embodiments, the microbial population comprises at least 80% of the distinct microbial species that are naturally present in the microbiota of kefir grains after culturing in milk.

[0154] Typically, the non-dairy composition comprises at least 5 distinct species of yeast, such as disclosed in the Example section of the specification.

[0155] The baseline kefir microbiota may include genera such as Lactobacillus, Leuconostoc, Lactococcus, Acetobacter, and Saccharomyces, among others.

[0156] The threshold of “at least 80%” may further include compositions containing at least 85%, at least 90%, at least 95%, or even 100% of the species present in the original kefir grains, as further described herein above.

[0157] The microbial population may be prepared by inoculating kefir grains into milk (bovine, goat, sheep, yak, camel), allowing fermentation to proceed under controlled conditions (e.g., 20–30°C for 12–48 hours) with no additional inoculum, harvesting the growing grain biomass, followed by washings to remove the dairy substrate. The resulting grain biomass may then be freeze-dried or otherwise stabilized before formulation.

[0158] In some embodiments, at least 50 %, 60 %, 70 %, 80 %, 90%, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or even 100 % of the microbes in the composition originate in the kefir grains when previously cultured in a dairy based product such as milk.

[0159] The composition may be formulated either:

[0160] (i) with the kefir grains themselves

[0161] (ii) without the grains.

[0162] In one embodiment, the kefir grains are removed, washed from the dairy product and incorporated directly into the final formulation (e.g., by homogenization or drying). This approach not only delivers the associated microbial population residing in the grains, but also provides a fermentable polysaccharide matrix that serves as a prebiotic. The grains include a matrix composed of polysaccharides such as kefiran, which support SCFA production, microbial colonization, enterocyte feeding and activity in the host gastrointestinal tract. This version may be presented as a paste, suspension, or encapsulated form. Such formulation can be regarded as symbiotic.

[0163] In another embodiment the kefir grains are removed and the microbiota is extracted and purified from the fermented / curdled milk.

[0164] In yet another embodiment, the crude kefir ferment, containing both grains and curdled milk is homogenized and the microbiota is purified and extracted from the mixed fraction.

[0165] Compositions expressed from the kefir grains, or from the curdled milk, or from both, are formulated for oral, endoscopic, colonoscopic or rectal delivery - as described herein above. In one embodiment, the formulation does not affect the relative abundance distribution of the microbial genera. Accordingly, in one embodiment, the relative abundance distribution of the microbial genera in the microbial population is substantially similar to the distribution of the microbial genera in the microbiota of the Kefir grains following fermentation.

[0166] The present inventors contemplate use of the presently disclosed microbial compositions for treating diseases associated with dysbiosis of the gut microbiome.

[0167] As used herein, the term “dysbiosis of the gut microbiome” refers to a state of imbalance or maladaptation in the composition, function, or spatial organization of the intestinal microbial community, relative to a healthy or homeostatic baseline. As opposed to eubiosis, dysbiosis may be characterized by one or more of the following features: a reduction in microbial diversity; a decrease in beneficial commensal taxa (e.g., Lactobacillus, Bifidobacterium);, an overrepresentation of potentially pathogenic or pro-inflammatory taxa (e.g., Enterobacteriaceae, Clostridium difficile);, an altered ratio of dominant phyla (e.g., Firmicutes to Bacteroidetes); disruptions in microbial metabolic output (e.g., short-chain fatty acids, bile acid conversion); or increased intestinal permeability and mucosal immune activation. Dysbiosis has been associated with a range of pathological conditions, including gastrointestinal, metabolic, neurological, immunological, and inflammatory diseases.

[0168] According to one embodiment, the disease is a metabolic disorder.

[0169] Examples of disorders contemplated by the present inventors include, but are not limited to obesity, type 2 diabetes (NIDDM), obesity, metabolic syndrome and metabolic associated fatty liver disease (MAFLD).

[0170] In another embodiment, the disease is a neurological or psychiatric disorder.

[0171] Examples of neurological or psychiatric disorders contemplated by the present inventors include, but are not limited to Autism, Parkinson’s disease (PD), depression and anxiety.

[0172] In another embodiment, the disease is a gastrointestinal disorder.

[0173] Examples of gastrointestinal disorders contemplated by the present inventors include, but are not limited to inflammatory bowel disease (IBD), irritable bowel syndrome (IBS) and colorectal cancer (CRC). In still another embodiment, the disease is an autoimmune and inflammatory disease.

[0174] Examples of autoimmune and inflammatory disease by the present inventors include, but are not limited to rheumatoid arthritis (RA), multiple sclerosis (MS), type I diabetes (IDDM), systemic lupus erythematosus (SLE), celiac disease and psoriasis. In yet another embodiment, the disease is cancer, whereby dysbiosis may lower the efficacy of immunotherapies, such as CARTs, Oncolytic Virotherapy, cytokines and Immune Checkpoint Blockade.

[0175] In certain embodiments, the compositions described herein are formulated as a food additive. In certain embodiments, the food additive disclosed herein further comprises other materials known in the art for inclusion in food additives, including, but not limited, water, purees, or other aqueous solutions, starch, binders, thickeners, colorants, flavorants, odorants, acidulants (e.g., lactic acid or malic acid, among others), vitamins, minerals, and combinations thereof. In certain embodiments, the food additive comprises between about 103and about 104CFU bacteria per gram of the food additive, between about 104and about 105CFU bacteria per gram of the food additive, between about 105and about 106CFU bacteria per gram of the food additive, between about 106and about 107CFU bacteria per gram of the food additive.

[0176] The present disclosure also provides a fortified food comprising the microbial compositions disclosed herein. In certain embodiments, the fortified food disclosed herein further comprises a base food. In certain embodiments, the food additive can be incorporated to a base food to form the fortified food. Any base foods known in the art can be used with the present disclosure. Nonlimiting examples of base foods include water, milk, fruit juices, vegetable juices, carbonated soft drinks, non-carbonated soft drinks, coffee, tea, beer, wine, liquor, alcoholic mixed drinks, bread, cakes, cookies, crackers, extruded snacks, soups, frozen desserts, fried foods, pasta products, potato products, rice products, corn products, fruit purees, wheat products, dairy products, confectionaries, hard candies, nutritional bars, breakfast cereals, bread dough, bread dough mix, sauces, processed meats, and even already fermented base foods such as kefir, kombucha, yakult, miso, natto, tempeh, kimchee, sauerkraut, cheeses, yogurt.

[0177] Administration of the microbial composition comprising mainly the bacteria, can be accomplished by any method likely to introduce the bacteria into the desired location. In certain embodiments, the composition can be administered to a subject, in the form of a food additive or a fortified food disclosed herein, by oral consumption. In certain embodiments, the bacteria can be mixed with a carrier and (for easier delivery to the digestive tract) be applied to liquid or solid food, feed, or drinking water. The carrier material should be non-toxic to the bacteria and the subject / patient. In certain embodiments, the carrier contains an ingredient that promotes viability of the bacteria during storage. The formulation can include added ingredients to improve palatability and improve shelf-life. Storage conditions before consumption may require cooling, to preserve microbial viability and also prevent further fermentation in the carrier. In certain embodiments, the carrier comprises a diluent, adjuvant, excipient, or vehicle with which the bacteria are administered. In certain embodiments, the carrier can be sterile liquids, such as water and oil emulsions, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. In certain embodiments, the carrier can be water or aqueous solution, saline solutions and aqueous dextrose and glycerol solutions. In certain embodiments, the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. Suitable carriers for therapeutic use are well known in the art and are described, for example, in “Remington’s Pharmaceutical Sciences” by E. W. Martin, and in “Remington: The Science and Practice of Pharmacy.” Lippincott Williams & Wilkins.

[0178] The choice of a carrier can be selected based on the intended route of administration and standard practice. In certain embodiments, oral, endoscopic, colonoscopic or rectal delivery can be used for delivery to the digestive tract. In certain embodiments, oral formulations comprise additional mixtures, such as milk, yogurt, purees and infant formula.

[0179] In certain embodiments, the duration and frequency of administration can vary between different subjects and is dependent upon the disease being treated.

[0180] In certain embodiments, solid dosages in the form of tablets or capsules are used for the delivery of the bacteria by mixing the bacteria with one or more components selected from the group consisting of sodium alginate, calcium carbonate, glyceryl monooleate, triethyl citrate, acetylated monoglyceride, and hypromellose acetate succinate (HPMCAS).

[0181] In certain embodiments, the bacteria or microbial compositions of the presently disclosed subject matter are formulated such that they can survive passage through the acidic environment of the stomach and such that they adjust quickly to the intestinal environment. Such formulation allows the presently described bacteria and microbial compositions to have an elongated half-life in the intestines.

[0182] In certain embodiments, prior to oral and certain cases of endoscopic administration, the subject may be pretreated with proton pump inhibitors (PPIs), to lower stomach acidity. This, however, might not be required in cases of formulations derived from naturally acidic fermented foods (such as kimchi, sauerkraut, kefir).

[0183] Prior to administration, the subject may be pretreated with an agent which reduces the number of naturally occurring microbes in the microbiome (e.g., by antibiotic treatment). According to a particular embodiment, the treatment significantly eliminates the naturally occurring gut microflora by at least 20 %, 30 % 40 %, 50 %, 60 %, 70 %, 80 % or even 90 %. In certain embodiments, the microbial compositions disclosed herein are administered to a subject who is healthy e.g., having a healthy BMI (e.g., 18.5-24.9). In certain embodiments, the probiotics or probiotic compositions disclosed herein are administered to a subject who has an overweight BMI (e.g., 25-29.9). In certain embodiments, the microbial compositions disclosed herein are administered to a subject who has a diagnosed disease. In certain embodiments, the microbial compositions are administered to the subject in the form of food additives or fortified foods disclosed herein. Dosage of the probiotic bacteria or probiotic composition disclosed herein for the subject can vary depending upon the characteristics of the subject (e.g., age, sex, ethnicity, weight, height, BMI, body fat percentage, and / or medical history), frequency of administration, manner of administration, clearance rate of the probiotic bacteria from the subject, and the like.

[0184] In certain embodiments, the initial dose can be larger, followed by smaller maintenance doses. In certain embodiments, the dose can be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered daily, semi- weekly, etc., to maintain an effective dosage level. In certain embodiments, a variety of doses are effective to achieve transient colonization of the gastrointestinal tract with the desired bacterial consortia in the formulation, for example and not by way of limitation, about 106CFU, about 107CFU, about 108CFU, about 109CFU, about 1010CFU, about 1011CFU, about 1012CFU, about 1013CFU, or about 1014CFU of probiotic bacteria can be administered in a single dose to a subject, depending on formulation method (e.g. wet, lyophilized). In certain embodiments, lower doses can also be effective, for example and not by way of limitation, about 104and about 105CFU of probiotic bacteria. In certain embodiments, the probiotic bacteria are administered to a subject in a dosage of between about 106and about 107CFU, between about 107and about 108CFU, between about 108and about 109CFU, between about 109and about IO10CFU, between about IO10and about 1011CFU, between about 1011and about 1012CFU, between about 1012and about 1013CFU, between about 1013and about 1014CFU. In certain embodiments, the probiotic bacteria are administered to a subject in a dosage of about 1010CFU of probiotics. In certain embodiments, the probiotic bacteria are administered to a subject in a dosage of up to about 1012CFU. In certain embodiments, the subject is a human. In certain embodiments, the subject is a domestic animal, e.g., a canine.

[0185] In some particular embodiments, appropriate doses or amounts to be administered may be extrapolated from dose-response curves derived from in vitro or animal model test systems. The effective dose or amount to be administered for a particular individual can be varied (e.g., increased or decreased) over time, depending on the needs of the individual. In some embodiments, it is the communal nature and interrelations between the taxa in the administered consortia that will be regarded, rather than the relative abundance of each taxa within the consortia. This relies on the premise that even rare taxa can efficiently and even preferentially colonize and amplify in the gut. In some embodiments, where bacteria are administered, an appropriate dosage comprises at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more bacterial cells. In some embodiments, the present invention encompasses the recognition that greater benefit may be achieved by providing numbers of bacterial cells greater than about 1000 or more (e.g., than about 1500, 2000, 2500, 3000, 35000, 4000, 4500, 5000, 5500, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, IxlO6, 2xl06, 3 xlO6, 4 xlO6, 5 xlO6, 6 xlO6, 7 xlO6, 8 xlO6, 9 xlO6, 1 xlO7, 1 xlO8, 1 xlO9, 1 xlO10, 1 xlO11, 1 xlO12, 1 xlO13or more bacteria.

[0186] In certain embodiments, when administered to a subject having a diagnosed disease, the optimal dosage can be empirically determined by treating physicians based on the stage of disease and patient statistics (e.g., age, height, weight, etc.). In certain embodiments, when administered to a subject who has a healthy BMI, or an overweight BMI, the optimal dosage can be empirically determined by the subject or a dietitian based on the subject’s statistics, e.g., age, sex, race, height, weight, BMI, body fat percentage, and / or medical history.

[0187] In certain embodiments, the microbial compositions disclosed herein can be delivered every 4, 12, 24, 36, 48, 60, or 72 hours. In certain embodiments, the microbial composition can be delivered with at least one second pharmaceutically active ingredient, where the second pharmaceutically active ingredient can be delivered simultaneously or sequentially (e.g., within a 4, 12, 24-hour or 1-week period) with the microbial composition. In certain embodiments, the microbial composition can be delivered with two, three, four, five, or six second pharmaceutically active ingredients. In certain embodiments, the treatment can last for at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 2 months, at least about 3 months, at least about 6 months, or at least about 1 year.

[0188] In certain embodiments, one or more preparations of different microbial compositions can be administered simultaneously (i.e., from one or more food source or combined different food sources) or sequentially (including administering at different times).

[0189] In certain embodiments, the present disclosure provides microbial compositions comprising bacteria at a concentration of between about 1 weight % and about 100 weight % (%w / w) of the microbial compositions. In certain embodiments, the bacteria are at a concentration of between about 1 ppm and about 1,000,000 ppm of the microbial compositions.

[0190] Compositions of some embodiments of the invention may be subject to food safety regulations. Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA or EMA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U. S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for safe treatment of an indicated condition, as is further detailed above.

[0191] Since bacteria are contemplated for health maintenance, and not necessarily for treatment of a disease, the presently disclosed microbial compositions are also contemplated for use as a health promoting agent is healthy subjects.

[0192] The present inventors further contemplate that the presently disclosed microbial composition are advantageous for treating a subject following antibiotic administration. It is conceived that administration of the composition reduces gastrointestinal side effects associated with antibiotic therapy and protects against further opportunistic colonization by pathogens, many of which have become antibiotic -resistant.

[0193] Thus, according to another aspect of the present invention, there is provided a method for restoring gut microbial diversity in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition described herein following antibiotic treatment.

[0194] Typically, the composition is administered within 1 to 14 days following completion of antibiotic therapy.

[0195] In one embodiment, the disease for which the antibiotic is used is a bacterial disease.

[0196] In particular, the disease is an infection of the gastrointestinal tract (e.g., caused by the bacterium Clostridium Difficile (C. Diff)). This has been noted as a frequent and severe contributor to nosocomial (hospital acquired) infections of the Gastrointestinal Tract (GIT), following antibiotics. C. Diff bacteria can also transform into a dormant resilient phase, by producing endospores, thus bringing about multiple cycles of recurrent C. Diff infection (rCDI).

[0197] In another embodiment, the disease is not a bacterial disease. In one embodiment, the disease is chronic. In another embodiment, the disease is acute. In another embodiment, it is rather a clinical condition, such as SIBO (Small Intestinal Bacterial Overgrowth), or SIFO (Small Intestinal Fungal Overgrowth).

[0198] Examples of diseases which may be treated using antibiotics include but are not limited to acne, appendicitis, atrial septal defect, bacterial arthritis, bacterial vaginosis, balance disorder, Bartholin's cyst, bursitis, pressure ulcer, bronchitis, conductive hearing loss, croup, cystic fibrosis, granuloma inguinale, duodenitis, dermatitis, emphysema, endocarditis, enteritis, gastritis, glomerulonephritis, gonorrhea, cardiovascular disease, Hidradenitis suppurativa, laryngitis, livedo reticularis, lymphogranuloma venereum, marasmus, mastoiditis, meningitis, myocarditis, nephrotic syndrome, Neurogenic bladder dysfunction, Non-gonococcal urethritis, Noonan syndrome, osteomyelitis, onychocryptosis, otitis externa, otitis media, Patent ductus arteriosus, pelvic inflammatory disease, perforated eardrum, pericarditis, peritonitis, pharyngitis, pilonidal cyst, pleurisy, prepatellar bursitis, pyelonephritis, sepsis, Stevens-Johnson syndrome, streptococcal pharyngitis, syphilis, tonsillitis, trichomoniasis, tuberculosis, ureterocele, urethral syndrome, urethritis, urinary tract infection and vertigo.

[0199] Examples of antibiotics contemplated by the present invention include, but are not limited to Daptomycin; Gemifloxacin; Telavancin; Ceftaroline; Fidaxomicin; Amoxicillin; Ampicillin; Bacampicillin; Carbenicillin; Cloxacillin; Dicloxacillin; Flucioxacillin; Mezlocillin; Nafcillin; Oxacillin; Penicillin G; Penicillin V; Piperacillin; Pivampicillin; Pivmecillinam; Ticarcillin; Aztreonam; Imipenem; Doripenem; Meropenem; Ertapenem; Clindamycin; Lincomycin; Pristinamycin; Quinupristin; Cefacetrile (cephacetrile); Cefadroxil (cefadroxyl); Cefalexin (cephalexin); Cefaloglycin (cephaloglycin); Cefalonium (cephalonium); Cefaloridine (cephaloradine); Cefalotin (cephalothin); Cefapirin (cephapirin); Cefatrizine; Cefazaflur; Cefazedone; Cefazolin (cephazolin); Cefradine (cephradine); Cefroxadine; Ceftezole; Cefaclor; Cefamandole; Cefmetazole; Cefonicid; Cefotetan; Cefoxitin; Cefprozil (cefproxil); Cefuroxime; Cefuzonam; Cefcapene; Cefdaloxime; Cefdinir; Cefditoren; Cefetamet; Cefixime; Cefmenoxime; Cefodizime; Cefotaxime; Cefpimizole; Cefpodoxime; Cefteram; Ceftibuten; Ceftiofur; Ceftiolene; Ceftizoxime; Ceftriaxone; Cefoperazone; Ceftazidime; Cefclidine; Cefepime; Cefluprenam; Cefoselis; Cefozopran; Cefpirome; Cefquinome; Fifth Generation; Ceftobiprole; Ceftaroline; Not Classified; Cefaclomezine; Cefaloram; Cefaparole; Cefcanel; Cefedrolor; Cefempidone; Cefetrizole; Cefivitril; Cefmatilen; Cefmepidium; Cefovecin; Cefoxazole; Cefrotil; Cefsumide; Cefuracetime; Ceftioxide; Azithromycin; Erythromycin; Clarithromycin; Dirithromycin; Roxithromycin; Telithromycin; Amikacin; Gentamicin; Kanamycin; Neomycin; Netilmicin; Paromomycin; Streptomycin; Tobramycin; Flumequine; Nalidixic acid; Oxolinic acid; Piromidic acid; Pipemidic acid; Rosoxacin; Ciprofloxacin; Enoxacin; Lomefloxacin; Nadifloxacin; Norfloxacin; Ofloxacin; Pefloxacin; Rufloxacin; Balofloxacin; Gatifloxacin; Grepafloxacin; Levofloxacin; Moxifloxacin; Pazufloxacin; Sparfloxacin; Temafloxacin; Tosufloxacin; Besifloxacin; Clinafloxacin; Gemifloxacin; Sitafloxacin; Trovafloxacin; Prulifloxacin; Sulfamethizole; Sulfamethoxazole; Sulfisoxazole; Trimethoprim-Sulfamethoxazole; Demeclocy cline; Doxycycline; Minocycline; Oxytetracycline; Tetracycline; Tigecycline; Chloramphenicol; Metronidazole; Tinidazole; Nitrofurantoin; Vancomycin; Teicoplanin; Telavancin; Linezolid; Cycloserine 2; Rifampin; Rifabutin; Rifapentine; Bacitracin; Polymyxin B; Viomycin; Capreomycin.

[0200] The present invention further contemplates using the compositions as adjuvants for treating a subject having a disease for which immunotherapy is therapeutically beneficial.

[0201] As used herein, the term “immunotherapy” refers broadly to any therapeutic intervention that modulates, enhances, suppresses, or redirects the immune system in order to prevent, treat, or ameliorate disease. Immunotherapy may be applied in the treatment of cancers, autoimmune diseases, infectious diseases, allergies, and chronic inflammatory conditions.

[0202] Immunotherapeutic agents suitable for use in the methods described herein may include, without limitation:

[0203] Immune Checkpoint Inhibitors:

[0204] These are monoclonal antibodies that block inhibitory pathways used by tumors or pathogens to evade immune detection. Examples include Anti-PD-1 antibodies (e.g., nivolumab, pembrolizumab); Anti-PD-Ll antibodies (e.g., atezolizumab, durvalumab, avelumab); Anti-CTLA-4 antibodies (e.g., ipilimumab) and Anti-TIGIT, anti-LAG3, or anti-TIM-3 antibodies under investigation

[0205] Cytokine Therapies:

[0206] These involve the administration of immune-modulating cytokines to stimulate immune cell activation and proliferation. Examples include Interleukin-2 (IL-2), including high-dose IL-2 and engineered variants (e.g., aldesleukin); Interleukin- 15 (IL-15) and its superagonists, Interferon-alpha (IFN-a) and interferon-gamma (IFN-y) and Tumor necrosis factor-alpha (TNF-a) agonists.

[0207] Cancer Vaccines:

[0208] These aim to induce a tumor- specific immune response. They may include peptide-based vaccines, dendritic cell vaccines, neoantigen-based formulations, and whole-cell tumor vaccines. Examples include Sipuleucel-T (Provenge), a dendritic cell vaccine approved for prostate cancer Adoptive Cell Therapies (ACT):

[0209] This includes therapies where immune cells are collected, modified or expanded ex vivo, and reinfused into the patient. Examples include CAR-T cell therapies (e.g., tisagenlecleucel, axicabtagene ciloleucel); Tumor-infiltrating lymphocytes (TILs) and TCR-engineered T cells targeting specific tumor antigens.

[0210] Immunomodulatory Antibodies:

[0211] These may target costimulatory receptors (agonists) or suppressive receptors (antagonists) to modulate immune responses. Examples include Anti-CD137 (4-1BB), anti-OX40, anti-CD40 agonists and Anti-CD47 or anti-SIRPa blocking agents to enhance phagocytosis of tumor cells.

[0212] Toll-Like Receptor (TLR) Agonists and Other Innate Immune Modulators:

[0213] These include agents that activate innate immune pathways to stimulate antigen presentation and T cell priming Examples include CpG oligodeoxynucleotides (TLR9), imiquimod (TLR7) and STING agonists.

[0214] The choice of immunotherapy agent may be determined by the specific disease indication, the patient’s immunological profile, tumor mutational burden (TMB), micro satellite instability (MSI) status, or other biomarkers predictive of response. The combination with the bacterial composition is intended to synergistically enhance efficacy, reduce resistance, or mitigate adverse immune-related events.

[0215] As used herein the term “about” refers to ± 10 %.

[0216] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0217] The term “consisting of’ means “including and limited to”.

[0218] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0219] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0220] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0221] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0222] As used herein, the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0223] As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

[0224] When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, sequencing depth or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

[0225] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0226] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

[0227] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

[0228] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. General references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader.

[0229] MATERIALS AND METHODS

[0230] Ferments and derived microbiomes

[0231] Two fermented food sources were analyzed:

[0232] Kefir grain ferment

[0233] A 1:5 ratio of Kefir Grains, to store bought 3% fat cow’s milk (pasteurized, non-UHT) was incubated at room temp (25 °C) for 24 hours. The Kefir Grains were strained and washed twice with saline. Kefir Grains were then mixed with saline at 1:1 ratio (w / w), homogenized and further analyzed by metagenomics.

[0234] Sauerkraut ferment

[0235] The following w / w ratios of ingredients were combined: 5: 2: 1: 1: 3 of shredded cabbage: water: grated carrot: grated onion: apple-mango-puree. Coarse kosher salt was added to a final concentration of 2% w / w. Floating cabbage mass originating from fermentation gas was daily pushed to the bottom of vessel. Duration of fermentation was 6 days at 25°C, final pH 3-4. The ferment was then further shredded in a blender and sieved. The filtrate was spun and washed twice with saline before metagenomic analysis.

[0236] In another embodiment, the microbiome was separated from the food source, by using a 63 micron stomacher bag. Further to that, the microbiome is washed twice and concentrated by centrifugation or density gradient.

[0237] Metagenomics

[0238] DNA extraction

[0239] DNA was extracted from samples using 270 µl GT lysis buffer and 30 µl proteinase K (from MagCore Genomic DNA Tissue Kit) along with 200 µl sample in bead beating tubes type C (GeneAid). Bead beating was performed for 2 minutes using a Biospec machine. Samples were then incubated at 60 degrees for 2 hours, and extracted on MagCore machine (RBC Bioscience) using MagCore Genomic DNA Tissue Kit cartridges and protocol. PCR1

[0240] 2 µl of DNA was used as template for initial PCR. Amplification was performed using PCRbio Hot start ready mix, using custom primers from Earth Microbiome Project containing CS1 / CS2 adaptors for 25 cycles in a volume of 25 µl.

[0241] PCR2

[0242] 2 pl sample from PCR1 amplified sample containing CS1 / CS2 adaptors was amplified for 10 cycles in 10 pl using Fluidigm Access Array Barcode library according to manufacturer’s protocol (2ul barcode per reaction). DNA was purified using Kapa Pure Beads at a ratio of 0.65X and quantified with qubit using Denovix DsDNA high sensitivity assay. DNA size and integrity was quantified by Tapestation using Agilent DNA screen tape and reagents.

[0243] Sequencing

[0244] Samples were run on a dedicated Miseq (Illumina) machine with 30% PhiX using MiSeq Reagent Kit v2 500PE. Demultiplexing was performed using bcl2fastq with default parameters allowing for 0 mismatches. Data was then mapped to PhiX using bowtie2 to remove PhiX control and unmapped reads were quantified, collected and examined using fastQC.

[0245] Analysis

[0246] Demultiplexed reads were uploaded into CLC genomics workbench (Qiagen), and analyzed using their 16S microbiome pipeline. The analysis workflow consisting of quality filtration of the sequence data, and operational taxonomic unit (OTU) clustering was performed with default parameter settings. The adaptor sequence was removed and the reads with a quality score lower than 25 or length <150 were discarded. The maximum number of acceptable ambiguous nucleotides was set to 2 and the length of the reads was fixed at 200-500bp. Chimeric sequences and singletons were detected and discarded. The remaining unique reads were used for OTU clustering, which was performed by alignment to the SILVA database at 97% sequence similarity.

[0247] RESULTS

[0248] Table 1 summarizes the species of bacteria identified in Kefir grains and Sauerkraut, as determined based on r16S NGS. Table 1

[0249] Species table in Kefir Grains and Sauerkraut

[0250] Species Species Taxonomy by r16S NGS Kefir

[0251] # Grains Sauerkraut Relative Relative Abundance Abundance (%) (%) 1 Bacteria; Actinobacteria; Actinobacteria; Actinomycetales; 0.020 0 Actinomycetaceae; Actinomyces; Actinomyces sp. oral clone EP053

[0252] 2 Bacteria; Actinobacteria; Actinobacteria; Actinomycetales; 0.015 0

[0253] Actinomycetaceae; Actinomyces; Uncultured bacterium

[0254] 3 Bacteria; Actinobacteria; Actinobacteria; Corynebacteriales; 0.015 0 Corynebacteriaceae; Corynebacterium; Uncultured bacterium

[0255] 4 Bacteria; Actinobacteria; Actinobacteria; Micrococcales; 0.033 0

[0256] Micrococcaceae; Rothia; Rothia dentocariosa

[0257] 5 Bacteria; Actinobacteria; Actinobacteria; Micrococcales; 0.005 0

[0258] Micrococcaceae; Rothia; Uncultured bacterium

[0259] 6 Bacteria; Actinobacteria; Actinobacteria; Propionibacteriales; 0.089 0 Propionibacteriaceae; Pseudopropionibacterium; Uncultured

[0260] bacterium

[0261] 7 Bacteria; Bacteroidetes; Bacteroidia; Bacteroidales; 0.015 0 Porphyromonadaceae; Porphyromonas; Uncultured bacterium

[0262] 8 Bacteria; Bacteroidetes; Bacteroidia; Bacteroidales; Tannerellaceae; 0.005 0

[0263] Tannerella; Ambiguous_taxa

[0264] 9 Bacteria; Bacteroidetes; Bacteroidia; Flavobacteriales; 0.023 0 Flavobacteriaceae; Capnocytophaga; Uncultured bacterium

[0265] 10 Bacteria; Bacteroidetes; Bacteroidia; Flavobacteriales; 0.033 0

[0266] Flavobacteriaceae; Capnocytophaga; Unidentified

[0267] 11 Bacteria; Epsilonbacteraeota; Campylobacteria; Campylobacterales; 0.010 0 Campylobacteraceae; Campylobacter; Uncultured bacterium

[0268] 12 Bacteria; Firmicutes; Bacilli; Lactobacillales; Aerococcaceae; 0.013 0

[0269] Abiotrophia; Unidentified

[0270] 13 Bacteria; Firmicutes; Bacilli; Lactobacillales; Lactobacillaceae; 0 0.019

[0271] Lactobacillus;

[0272] 14 Bacteria; Firmicutes; Bacilli; Lactobacillales; Lactobacillaceae; 0.744 0.056

[0273] Lactobacillus; Ambiguous_taxa

[0274] 15 Bacteria; Firmicutes; Bacilli; Lactobacillales; Lactobacillaceae; 0 16.0

[0275] Lactobacillus; Lactobacillus brevis

[0276] 16 Bacteria; Firmicutes; Bacilli; Lactobacillales; Lactobacillaceae; 0 0.037

[0277] Lactobacillus; Lactobacillus fuchuensis

[0278] 17 Bacteria; Firmicutes; Bacilli; Lactobacillales; Lactobacillaceae; 96.9 0

[0279] Lactobacillus; Lactobacillus kefiranofaciens ZW3

[0280] 18 Bacteria; Firmicutes; Bacilli; Lactobacillales; Lactobacillaceae; 0 0.879

[0281] Lactobacillus; Lactobacillus pentosus

[0282] 19 Bacteria; Firmicutes; Bacilli; Lactobacillales; Lactobacillaceae; 0 0.206

[0283] Lactobacillus; Lactobacillus plantarum

[0284] 20 Bacteria; Firmicutes; Bacilli; Lactobacillales; Lactobacillaceae; 0 0.711

[0285] Lactobacillus; Lactobacillus sp. Lu5

[0286] 21 Bacteria; Firmicutes; Bacilli; Lactobacillales; Lactobacillaceae; 0.010 0

[0287] Lactobacillus; Uncultured Bacillus sp

[0288] 22 Bacteria; Firmicutes; Bacilli; Lactobacillales; Lactobacillaceae; 0.020 0.112

[0289]

[0290] Lactobacillus; Uncultured bacterium Bacteria; Firmicutes; Bacilli; Lactobacillales; Streptococcaceae; 0 0.056 Lactococcus;

[0291] Bacteria; Firmicutes; Bacilli; Lactobacillales; Streptococcaceae; 0 0.898

[0292] Lactococcus; Ambiguous_taxa

[0293] Bacteria; Firmicutes; Bacilli; Lactobacillales; Streptococcaceae; 0.005 0.823

[0294] Lactococcus; Lactococcus lactis

[0295] Bacteria; Firmicutes; Bacilli; Lactobacillales; Streptococcaceae; 0.176 79.2 Lactococcus; Lactococcus lactis subsp. lactis

[0296] Bacteria; Firmicutes; Bacilli; Lactobacillales; Streptococcaceae; 0 0.861

[0297] Lactococcus; Uncultured bacterium

[0298] Bacteria; Firmicutes; Bacilli; Lactobacillales; Streptococcaceae; 0.010 0

[0299] Streptococcus; Streptococcus sanguinis

[0300] Bacteria; Firmicutes; Bacilli; Lactobacillales; Streptococcaceae; 1.330 0

[0301] Streptococcus; Uncultured bacterium

[0302] Bacteria; Firmicutes; Bacilli; Lactobacillales; Streptococcaceae; 0.005 0

[0303] Streptococcus; Uncultured Streptococcus sp

[0304] Bacteria; Firmicutes; Clostridia; Clostridiales; Lachnospiraceae; 0.051 0

[0305] Blautia; Uncultured bacterium

[0306] Bacteria; Firmicutes; Negativicutes; Selenomonadales; 0.005 0 Veillonellaceae; Veillonella; Ambiguous_taxa

[0307] Bacteria; Fusobacteria; Fusobacteriia; Fusobacteriales; 0.015 0 Fusobacteriaceae; Fusobacterium; Ambiguous_taxa

[0308] Bacteria; Fusobacteria; Fusobacteriia; Fusobacteriales; 0.005 0 Fusobacteriaceae; Fusobacterium; Uncultured bacterium

[0309] Bacteria; Fusobacteria; Fusobacteriia; Fusobacteriales; 0.010 0 Leptotrichiaceae; Leptotrichia; Ambiguous_taxa

[0310] Bacteria; Fusobacteria; Fusobacteriia; Fusobacteriales; 0.005 0 Leptotrichiaceae; Leptotrichia; Uncultured bacterium

[0311] Bacteria; Fusobacteria; Fusobacteriia; Fusobacteriales; 0.005 0 Leptotrichiaceae; Leptotrichia; Uncultured Leptotrichia sp

[0312] Bacteria; Proteobacteria; Gammaproteobacteria; 0.005 0 Betaproteobacteriales; Burkholderiaceae; Lautropia;

[0313] Bacteria; Proteobacteria; Gammaproteobacteria; 0.036 0 Betaproteobacteriales; Burkholderiaceae; Lautropia; Uncultured

[0314] bacterium

[0315] Bacteria; Proteobacteria; Gammaproteobacteria; 0.031 0 Betaproteobacteriales; Burkholderiaceae; Lautropia; Unidentified

[0316] Bacteria; Proteobacteria; Gammaproteobacteria; 0.023 0 Betaproteobacteriales; Neisseriaceae; Eikenella; Uncultured

[0317] bacterium

[0318] Bacteria; Proteobacteria; Gammaproteobacteria; 0.015 0 Betaproteobacteriales; Neisseriaceae; Neisseria; Kingella potus

[0319] Bacteria; Proteobacteria; Gammaproteobacteria; 0.015 0 Betaproteobacteriales; Neisseriaceae; Neisseria; Neisseria elongata

[0320] subsp. glycolytica ATCC 29315

[0321] Bacteria; Proteobacteria; Gammaproteobacteria; 0.084 0 Betaproteobacteriales; Neisseriaceae; Neisseria; Uncultured

[0322] bacterium

[0323] Bacteria; Proteobacteria; Gammaproteobacteria; 0.005 0 Betaproteobacteriales; Neisseriaceae; Neisseria; Uncultured

[0324] Neisseria sp

[0325] Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacteriales; 0.143 0 Enterobacteriaceae; Citrobacter; Ambiguous_taxa

[0326] Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacteriales; 0.051 0.019

[0327]

[0328] Enterobacteriaceae; Enterobacter; Ambiguous_taxa 48 Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacteriales; 0 0.094 Enterobacteriaceae; Enterobacter; Enterobacter sp. UCD- UG_FMILLET

[0329] 49 Bacteria; Proteobacteria; Gammaproteobacteria; Pasteurellales; 0.046 0

[0330] Pasteurellaceae; Haemophilus simiae; Haemophilus simiae

[0331] 50 Bacteria; Proteobacteria; Gammaproteobacteria; Pasteurellales; 0.010 0

[0332] Pasteurellaceae; Haemophilus; Uncultured bacterium

[0333] Number of species: 40 15 Shannon Diversity Index: 0.21 0.72 Bray-Curtis Dissimilarity: 0.99 Number of overlapping species: 5 Number of unique species: 50

[0334]

[0335] In total, 40 different species of bacteria were identified in Kefir grains and 15 different species of bacteria were identified in the Sauerkraut. The Shannon Diversity for the Kekir Grains is 0.21 and for the Sauerkraut 0.72. The Bray-Curtis Dissimilarity between the Kefir Grains and the Sauerkraut is 0.99 (min=0, max=1), alluding to a marked dissimilarity between the two ferments. It is exactly this dissimilarity that enables diversity upon combining different food ferments into one composition. Similarly, the overall Shannon diversity index of a composition can be increased by modulating the assortment of combined microbiota derived from different food ferments.

[0336] Additionally, at least five species of yeast were identified in Kefir grains, as determined by r18S NGS, including Kluyveromyces, Saccharomyces and Vanderwaltozyma.

[0337] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0338] All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety.

Claims

WHAT IS CLAIMED IS:

1. A method of generating a microbial composition comprising:(a) culturing at least one food source under conditions that promote native microbial growth; (b) separating resulting microbes from the cultured food to obtain a composition comprising at least 80% of the distinct microbial species present in the microbiota of the cultured food; and (c) processing the microbes by formulating the composition for oral, endoscopic, colonoscopic or rectal administration.

2. The method of claim 1, wherein the microbes are formulated as a capsule, as a tablet, as a dry powder, as a suppository or as a suspension.

3. The method of claim 1, wherein die processing comprises freezing, drying or homogenization.

4. The method of claims 1 or 2, wherein the culturing is affected at a pH level that inhibits the growth of pathogenic microorganisms.

5. The method of claims 1 or 4, wherein the separation step comprises filtering, centrifugation, or decantation.

6. The method of any one of claim 1-5, wherein the at least one food source is a lactic acid fermentable food substrate.

7. The method of any one of claims 1-6, wherein the cultured food is a plant-based food, algae, an animal-based food or kefir grains.

8. A composition comprising microbes, wherein the composition is generated according to the method of any one of claims 1-7.

9. The composition of claim 8, wherein said microbes are viable.

10. The composition of claims 8 or 9, wherein at least 80 % of the resulting microbes in the composition originate from the fermented food source.

11. The composition of any one of claims 8-10, being non-caloric.

12. The composition of any one of claims 8-11, wherein the relative abundance distribution of the microbial species in the composition is substantially similar to the distribution of the microbial species in the microbiota of the food source following said culturing.

13. A non-caloric composition comprising a microbial population that comprises at least 80% of the distinct microbial species that are present in the microbiota of at least one fermented food; the composition being formulated for oral, endoscopic, colonoscopic or rectal administration.

14. The non-caloric compositions of claim 13, wherein at least 50 % of the microbes of the non-caloric composition originate from the food source.

15. A non-caloric composition comprising a microbial population that comprises at least 10 distinct microbial species present in the microbiota of a fermented food, the composition being formulated for oral, endoscopic, colonoscopic or rectal administration.

16. The non-caloric composition of claim 15, wherein said 10 distinct microbial species originate in the food source of the fermented food.

17. The non-caloric composition of claims 15 or 16, wherein at least 50 % of the microbes of the non-caloric composition originate from the food source.

18. The non-caloric compositions of any one of claims 13-15, wherein said composition is formulated as a capsule, a tablet, dry powder, a suspension or a suppository.

19. The non-caloric composition of any one of claims 13-18, wherein the relative abundance distribution of the microbial genera in the microbial population of the fermented composition is substantially similar to the distribution of the microbial genera in the microbiota of the food source following culturing.

20. The non-caloric composition of any one of claims 13-19, wherein said food source is a natural food source.

21. The non-caloric composition of any one of claims 13-20, wherein said food source is a lactic acid fermentable food substrate.

22. The non-caloric composition of claim 20 or 21, wherein said food source is a plant.

23. The non-caloric composition of claim 20 or 21, wherein said food source comprises a fruit or a vegetable.

24. The non-caloric composition of claim 23, wherein said vegetable comprises a cruciferous vegetable.

25. The non-caloric composition of claim 20 or 21, wherein said food source comprises Kefir grain.

26. The non-caloric composition of any one of claims 13-25, wherein said microbial population comprises bacteria.

27. The non-caloric composition of any one of claims 13-26, wherein microbes of the microbial population are viable.

28. The non-caloric composition of claim 13, comprising at least 10 different species of bacteria present in the microbiota of the same fermented food.

29. The non-caloric composition of claims 15 or 28, comprising at least 20 different species of bacteria present in the microbiota of the same fermented food.

30. A non-dairy composition comprising a microbial population that comprises at least 80% of the distinct microbial species that are naturally present in the microbiota of Kefir grains, wherein at least 50 % of the microbes of said non-dairy composition originate from the Kefir grains when cultured in milk, the composition being formulated for oral, endoscopic, colonoscopic or rectal administration.

31. The non-dairy composition of claim 30, wherein the composition is formulated as a capsule, a tablet, a dry powder, a suspension or a suppository'.

32. The non-dairy composition of claim 30, wherein the composition is homogenized, dried or frozen.

33. The non-dairy composition of claims 30 or 31, wherein the relative abundance distribution of the microbial species in the microbial population is substantially similar to the distribution of the microbial species in the microbiota of the Kefir grains following fermentation.

34. The non-dairy composition of any one of claims 30-33, comprising kefir grains washed away from milk.

35. The non-dairy composition of claim 30, wherein microbes of said microbial population are viable.

36. The non-dairy composition of claim 30, comprising at least 5 distinct species of yeast.

37. The non-dairy composition of claims 30 or 36, comprising at least 20 distinct species of bacteria.

38. The composition of any one of claims 13-37, being devoid of fecal material.

39. The composition of any one of claims 13-37, being devoid of pathogenic taxa.

40. A fortified food comprising the composition of any one of claims 8-39.

41. The composition or fortified food of any one of claims 13-40, for use in treating a disease associated with dysbiosis.

42. A method of treating a subject having a disease associated with dysbiosis of the gut microbiome comprising administering to the subject a therapeutically effective amount of the composition or fortified food of any one of claims 13-40, thereby treating the subject.

43. The composition, fortified food or method of claims 41 or 42, wherein the disease is a metabolic disorder.

44. The composition, fortified food, or method of claim 43, wherein the metabolic disorder is selected from the group consisting of obesity, metabolic syndrome, type 2 diabetes (NIDDM) and metabolic associated fatty liver disease (MAFLD).

45. The composition, fortified food or method of claims 40, 41 or 42, wherein the disease is a neurological or psychiatric disorder.

46. The composition, fortified food or method of claims 40, 41 or 42, wherein the disease is a gastrointestinal disorder.

47. The composition, fortified food or method of claim 46, wherein said gastrointestinal disorder is selected from the group consisting of inflammatory bowel disease (IBD), irritable bowel syndrome (IBS) and colorectal cancer (CRC).

48. The composition, fortified food or method of claims 40, 41 or 42, wherein said disease is an autoimmune and inflammatory disease.

49. The composition, fortified food or method of claim 48, wherein said autoimmune and inflammatory disease is selected from the group consisting of rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), celiac disease and psoriasis.

50. A method for restoring gut microbial diversity in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of any one of claims 13-39 following antibiotic treatment.

51. The composition of any one of claims 13-39, for use in restoring gut microbial diversity following antibiotic treatment.

52. The method or composition of claims 50 or 51, wherein the composition is administered by the oral, endoscopic, colonoscopic or rectal route.

53. The method or composition of any one of claims 50-52, wherein the composition is administered within 1 to 14 days following completion of antibiotic therapy.

54. The method of any one of claims 50-53, wherein administration of the composition reduces gastrointestinal side effects associated with antibiotic therapy.

55. A method of maintaining the health of a subject comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 13-40, thereby maintaining the health of the subject.

56. The method of any one of claims 42-55, wherein the administering comprises orally administering.

57. A method of treating a subject having a disease for which immunotherapy is therapeutically beneficial, comprising administering to the subject a therapeutically effective amount of the immunotherapy and the composition of any one of claims 13-40, thereby treating the subject.

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