Deconstructed Soil

Abstract

The present invention includes a defined, safe soil substitute, referred to herein as "deconstructed soil," that supports ecological balance within anaerobic microbial ecosystems, such as those in the human gut, by shifting energy utilization rates to favor the growth of bacteria associated with gut health and away from the most dominant and presumed harmful species. The invention allows vulnerable bacterial populations to recover from apparent extinction from the ecosystem in question. Because deconstructed soil does not contain any prebiotic or probiotic ingredients, it represents a novel product concept for preventing and treating conditions associated with dysfunction of microbial ecosystems in the digestive tracts of humans and animals, as well as other anaerobic microbial ecosystems.

Description

Technical Field

[0001] The present invention relates to deconstructed soil (DS) compositions in the regulation of microbial ecosystems by promoting the recovery of vulnerable bacterial species in the gut microbiota and favoring beneficial bacterial groups at the expense of species presumed to have a detrimental effect on health, and to their use.

Background Art

[0002] The spread of Western lifestyle diseases is most likely the result of modern living conditions that deviate significantly from the natural environment to which humans have adapted through biological evolution. Highly processed foods, aseptic environments, poorly understood hygiene practices, and lack of exposure to soil and other products in the natural environment can all be contributing factors. Such lifestyle diseases are associated with ecological disturbances within the gut microbiota, the so-called gut microbiome (GM). These ecological disturbances, often referred to as GM dysbiosis, have been associated with various disease states that represent a fundamental departure from the "one microbe - one disease" concept that has dominated medicine for generations. Therefore, attempts to prevent or cure diseases associated with GM dysbiosis must be based on an understanding of how extremely complex ecosystems such as the GM function and respond to food and other environmental inputs. The validity of the deconstructed soil described herein is that it modulates the GM by fundamental ecological principles and could thus be a preferred product category for combating lifestyle diseases in urbanized Western countries.

[0003] Biological Rationality of Geophagy The concept of DS as described herein can be better understood and evaluated when incorporated within a broader biological and evolutionary context. Geophagy, the spontaneous ingestion of soil, is widely observed among animal and human populations. In humans, within many cultures, the practice is particularly common among children as well as pregnant women. The biological rationale behind this behavior is not fully understood. However, since it is so widespread, it can be assumed to contribute to something beneficial. If it were dangerous or toxic, geophagy would have been abandoned by evolution.

[0004] Soil is a source of minerals. Thus, the spontaneous ingestion of soil is often regarded as an instinctive way to ensure an adequate supply of minerals and micronutrients. Geophagy is a normal human behavior, yet most people in Western societies view it as something that makes them feel queasy, strange, and abnormal. Thus, geophagy is not socially or mentally accepted as a health-promoting habit. Also, medical experts have not evaluated it. Even if it were accepted, in modern times it would not be recommended, especially in urbanized areas, due to the risks associated with contamination by heavy metals, toxic chemicals, and potential pathogens.

[0005] Exposure to soil has been shown to significantly modulate the GM of mice. In model experiments, mice raised on soil containing litter have been shown to have a reduced tendency to develop asthma. Additionally, repeated skin exposure to soil preparations has been associated with an increase in the diversity of microorganisms within the GM of healthy humans. High exposure to non-contaminated soil and components present in natural environments is also thought to contribute to a reduced incidence of non-communicable diseases in rural populations and hunter-gatherer communities. These effects are generally attributed to exposure to living microorganisms in these environments, but little attention has been paid to the abiotic components of soil, such as those within the DS presented herein. The DS product of the present invention corresponds to a sterilized, safe, and consistent substitute for exposure to natural soil.

[0006] Prior art In contrast to existing products that address GM dysbiosis, the DS described herein does not contain live microorganisms (probiotics), energy substrates for microorganisms (prebiotics), and metabolites left in the growth medium in which the microorganisms were cultured (postbiotics). The present invention takes into account the ecological complexity inherent in GM ecosystems by enhancing the energy distribution among the members of this community, rather than introducing live bacteria or supplying additional energy to a specific group of microorganisms within the community.

[0007] Probiotics aim to introduce a large number, one, or a few bacterial species into the GM. Probiotic bacteria, which are mostly lactic acid bacteria and are often foreign to the human gut, are generally considered beneficial. However, this view has little scientific support. In the case of species of the genus Lactobacillus, the most commonly used bacteria in probiotic preparations, each will usually carry indigenous species and strains that are already adapted to the host environment, but these usually appear in relatively low abundance in the human large intestine.

[0008] The microbiome is highly individual-specific, and isolating probiotic strains from diverse sources may not be an appropriate approach for introducing microorganisms that are presumed to be beneficial to hosts with different GM ecological environments. This is important because many of the health effects ascribed to probiotics are strain-specific. Unlike probiotics, the present invention presented herein does not introduce any live microorganisms, but helps to provide suitable conditions for indigenous bacteria to exist in a host adapted to them and coexist with other microorganisms within the same ecosystem.

[0009] Prebiotics as a concept were defined in 1995. And since then its definition has undergone many changes. According to the International Probiotics and Prebiotics Science Association (ISAPP), prebiotics are "substrates selectively utilized by host microorganisms conferring health benefits". The term "substrate" means a substance from which an organism obtains or can obtain its nutrients. Most prebiotics are oligosaccharides, which are growth substrates for bacteria that are presumed to be beneficial, mainly those belonging to the genera Lactobacillus and Bifidobacterium.

[0010] Since the DS of the present invention does not contain any energy substrates for the growth of bacteria under anaerobic conditions, its action on the GM ecological environment described in the following examples must be due to other mechanisms.

[0011] Postbiotics are low-molecular-weight microbial metabolites, including quorum-sensing molecules, that are produced during microbial growth and left in the culture medium. Such metabolites do not exist in the products of the present invention.

[0012] An alternative to the use of pro-, pre- and postbiotics for modulating GM is to transplant a complete microbial ecosystem in the form of fresh feces from a healthy donor, so-called fecal microbiota transplantation (FMT). FMT has been very successful in the treatment of antibiotic treatments associated with diarrhea caused by Clostridium difficile. These are bacteria that are normally present within a healthy GM, but in the extreme case of GM dysbiosis most often caused by antibiotics, it becomes a life-threatening toxin-producing bacterium. However, FMT is not a realistic alternative for the treatment of lifestyle diseases caused by dysbiotic GM, as it is a method outside the scope of the present invention.

[0013] In addition to the above categories of potential GM regulators, dietary fibers (both soluble and insoluble) have been investigated in this regard. Lentil fiber and fructooligosaccharides have been found to have some effect on members of a certain GM community, for example, reducing the abundance of Faecalibacterium prausnitzii. The reduction in the abundance of F. prausnitzii is in stark contrast to the observations made using the present invention.

[0014] Purified humic materials extracted from Leonardite-type geological deposits have been shown to be safe and without any adverse effects when administered orally in capsule form in a test conducted on 15 healthy volunteers (Non-Patent Document 1). This product appears to have the potential to modulate human GM, but the results presented in this test did not agree, lead to a conclusion, or were not statistically significant for most of the observations. This could be due to the small number of human volunteers in this pilot study and the fact that they were all healthy and presumably not GM-dysbiotic. There were also methodological limitations related to the microbiota analysis used in this test. For example, while no net increase in the Akkermansia genus was reported in this test, at the same time, a statistically significant increase in the abundance of Akkermansia muciniphila, the only species of the Akkermansia genus known to colonize the human intestine, was reported.

[0015] β-glucan is a family of β-D-glucose polysaccharides and is widely found in the cell walls of plants, fungi, and bacteria. Other previously described uses of β-glucan include its use as a drug delivery system safe for human use (Patent Document 1) and its use as a filler in animal feed. The β-glucan of barley and other grains is an unbranched mixed β-1,3 / 1,4-glucan, which is hydrolyzed by enzymes in the digestive tract and used by microorganisms as an energy substrate. Barley glucan and the hydrolyzates (oligomers) of barley glucan thus meet the definition of prebiotics. By this function, the β-glucan of grains including barley glucan provides nutrients and can thereby support the growth of the genera Lactobacillus and Bifidobacterium.

[0016] The human intestine does not secrete enzymes that hydrolyze β-1,3- or β-1,6-glycosidic bonds and particulate β-1,3 / 1,6-glucan products used as one of the components of DS. Therefore, it is resistant to enzymatic digestion in the digestive tract. It is also a very high-density structure that cannot be easily accessed by microorganisms in the anaerobic compartment of the intestinal tract, and observations from actual use suggest that the particles are excreted in feces. The particulate β-1,3 / 1,6-glucan component of the DS of the present invention has the technical advantage of forming a solid gel after autoclaving. Therefore, it corresponds to an ideal matrix that can incorporate other components of DS therein. This is important when formulating DS products not only for oral administration but also for local use in the treatment of wound infections or in dental care products.

Prior Art Documents

Patent Documents

[0017]

Patent Document 1

Non-Patent Documents

[0018] [Non-Patent Document 1] Swidsinski et al., 2017 [Summary of the Invention] [Problems to be Solved by the Invention]

[0019] The basic idea behind the concept of the product of the present invention is the opposite of that of prebiotics, that is, the product does not supply external energy to the GM ecosystem. Instead, in order to prevent and counteract dysbiosis, what DS aims at is to force the ecosystem to a mechanism in which individual species cooperate to share a limited energy supply. The β-1,3 / 1,6-glucan preparation used to illustrate the present invention has a chemical structure different from that of, for example, β-1,3 / 1,4-glucan found in Triticum aestivum, and is not utilized as an energy substrate by intestinal bacteria. It corresponds to a well-characterized β-glucan that is in the same chemical category as the β-glucan of soil fungi such as lentinan and has the same biological mechanism of action. The β-1,3 / 1,6-glucan used in the DS of the present invention acts as a substitute for the mycelial β-1,3 / 1,6-glucan found in natural forest soil.

[0020] The GM-regulating effect of DS may be argued to be merely the "action of fiber" obtained by the structure of any humus or the structure of lignin. We have demonstrated in the following examples that although both lignin and pure humic materials affect GM, their GM-regulating properties are completely different from those of DS. [Means for Solving the Problems]

[0021] The deconstructed soil (DS) composition of the present invention comprises two organic components and one inorganic material found in virgin soil, namely, 1) Humic materials (humus) containing black (oxidized) iron remaining after the microbiological and chemical decay of plant lignocellulosic materials in sediments or natural soils, 2) Fungal cell wall β-1,3 / 1,6-glucan, 3) Inorganic clay materials (illite), and contains.

[0022] The black humic material used in this study is not pure humic acid. It contains cellulose / β-glucan and more than 10% (dry weight basis) of inorganic matter that is normally present in soil, so it can be a reasonable substitute for forest soil. Still, we also made DS formulations enriched with inorganic soil materials such as clay (illite). Enrichment with β-glucan and clay minerals did not change the general microbiota-regulating properties of the basic DS composition, so we showed that it can be used when there is a need to modify the texture properties of DS.

[0023] The components of DS cannot be utilized as energy substrates for the growth of microorganisms under anoxic conditions. Still, DS has a significant effect on anaerobic or microaerophilic human GM, for example, in ecological environments such as the large intestine and cecum. It regulates the human GM by strongly and favorably influencing the growth of bacterial species associated with good health, unfavorably influencing the growth of bacterial groups presumed to have a bad effect on health, and promoting the recovery of species that are functionally important, rare, and fragile. This was quite unexpected because it could not have been predicted based on the current understanding of how anaerobic microbial ecosystems such as the human intestine function and how individual species within such ecosystems interact with each other.

[0024] DS does not fall within the definition of probiotics (living microorganisms), prebiotics (growth substrates for gut microbiota), or postbiotics (microbial metabolites). The DS of the present invention corresponds to an alternative concept for promoting healthy GM. We hypothesize that the DS creates a chemical environment and physical structure for the cooperative utilization of energy substrates that are otherwise not available to microorganisms in anaerobic and microaerophilic environments.

[0025] The DS composition according to the present invention containing black humic material and β-glucan is formulated for use in the regulation of anaerobic microbial ecosystems. (Appendix) The technical ideas that can be grasped from the embodiments and modified examples described herein are described below. [Item 1] A deconstructed soil composition containing black humic material and β-glucan. [Item 2] The composition according to item 1, wherein the β-glucan is resistant to microbial degradation. [Item 3] The composition according to items 1 and 2, wherein the β-glucan is a fungal type β-1,3 / 1,6-glucan. [Item 4] The composition according to item 3, wherein the β-glucan is a β-1,3 / 1,6-glucan obtained from yeast. [Item 5] The composition according to items 1 to 4, wherein the black humic material contains iron and lignocellulose. [Item 6] The composition according to any one of items 1 to 5, wherein the black humic material is of the Leonardite type. [Item 7] The composition according to any one of items 1 to 6, containing clay minerals. [Item 8] The composition according to any one of items 1 to 7, wherein the ratio of β-1,3 / 1,6-glucan to black humic material is in the range of 1:100 to 1:1, preferably 5:100. [Item 9] The composition according to item 8, wherein the ratio of β-1,3 / 1,6-glucan to the black humic material is 3.5:100. [Item 10] The composition according to any one of items 1 to 8, wherein the ratio of β-1,3 / 1,6-glucan to the black humic material to the clay mineral is in the range of 1:100:5 to 5:100:15. [Item 11] The composition according to item 10, wherein the ratio of β-1,3 / 1,6-glucan to the black humic material to the clay mineral is 3.5:100:10. [Item 12] The composition according to any one of items 1 to 11, for use in regulating an anaerobic microbial ecosystem. [Item 13] Use of the composition according to any one of items 1 to 12 in the treatment of dysbiosis in a target organism, wherein the target organism is selected from the group consisting of mammals, birds, and aquaculture species. [Item 14] The use according to item 13, wherein the mammal is a human. [Item 15] The use according to item 13, wherein the mammal is a pet or a livestock. [Item 16] The use according to item 13, wherein beneficial oxygen-sensitive intestinal bacteria are selectively favored under anaerobic and microaerophilic conditions. [Item 17] The use according to item 13, wherein harmful oxygen-sensitive intestinal bacteria are selectively disadvantaged under anaerobic and microaerophilic conditions. [Item 18] The use according to item 13, wherein the growth of Faecalibacterium prausnitzii is enhanced under anaerobic conditions. [Item 19] The use according to item 13, wherein the growth of Prevotella copri is enhanced under anaerobic conditions. [Item 20] The use according to item 13, wherein the growth of Akkermansia muciniphila is enhanced under anaerobic conditions. [Item 21] The use according to item 13, wherein the growth of Methanobrevibacter smithii is enhanced under anaerobic conditions. [Item 22] The use according to item 13, wherein the growth of the genus Bifidobacterium is enhanced under anaerobic conditions. [Item 23] The use according to item 13, wherein the growth of the genus Lactobacillus is enhanced under anaerobic conditions. [Item 24] The use according to item 13, wherein the growth of Faecalibacterium prausnitzii is enhanced under microaerophilic conditions. [Item 25] The use according to item 13, wherein the growth of Prevotella copri is enhanced under microaerophilic conditions. [Item 26] The use according to item 13, wherein the growth of Akkermansia muciniphila is enhanced under microaerophilic conditions. [Item 27] The use according to item 13, wherein the growth of Methanobrevibacter smithii is enhanced under microaerophilic conditions. [Item 28] The use according to item 13, wherein the growth of the genus Bifidobacterium is enhanced under microaerophilic conditions. [Item 29] The use according to item 13, wherein the growth of the genus Lactobacillus is enhanced under microaerophilic conditions. [Item 30] The use according to item 13, wherein the growth of Clostridium perfringens is disadvantaged under anaerobic conditions. [Item 31] The use according to item 13, wherein the growth of Finegoldia magna is disadvantaged under anaerobic conditions. [Item 32] The use according to item 13, wherein the growth of Alistipes shahii is disadvantaged under anaerobic conditions. [Item 33] The use according to item 13, wherein the growth of the genus Staphylococcus is disadvantaged under anaerobic conditions. [Item 34] Anaerobic conditions, bacteri RoyUse according to item 13 that is disadvantageous to the growth of Bacteroides umiformis. [Item 35] Under anaerobic conditions, bacteria Roy Use according to item 13 that is disadvantageous to the growth of Bacteroides vulgatus. [Item 36] Use according to item 13 that is disadvantageous to the growth of Clostridium perfringens under microaerophilic conditions. [Item 37] Use according to item 13 that is disadvantageous to the growth of Finegoldia magna under microaerophilic conditions. [Item 38] Use according to item 13 that is disadvantageous to the growth of Allistipes shahii under microaerophilic conditions. [Item 39] Use according to item 13 that is disadvantageous to the growth of Staphylococcus under microaerophilic conditions. [Item 40] Under microaerophilic conditions, bacteria Roy Use according to item 13 that is disadvantageous to the growth of Bacteroides uniformis. [Item 41] Under microaerophilic conditions, bacteria Roy Use according to item 13 that is disadvantageous to the growth of Bacteroides vulgatus.

Brief Description of Drawings

[0026]

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Mode for Carrying Out the Invention

[0027] Soil and Deconstructed Soil (DS) Natural forest soil consists of sandy clay as well as a mixture of dead plants, soil-dwelling animals, bacteria, and fungi that have been microbiologically and chemically decomposed. The surface of soil particles is usually exposed to oxygen and thus is an environment for aerobic soil microorganisms. Anaerobic species are limited to the oxygen-free sediment layer and the interior of individual soil particles. In an oxygen-free soil environment, microorganisms cannot use the lignin fragments in the plant's lignocellulose structure as an energy source, and the lignocellulose undergoes very slow chemical and microbiological modification reactions. Dead plants as well as soil bacteria and fungi thus remain, over very long periods, as a chemically extremely complex mixture of humic materials (derived from plant lignin), polysaccharides (derived from plant cell walls), and β-1,3 / 1,6-glucan (derived from fungal cell walls). The recent plant material in sediments and soil is rapidly replaced, but when the humic material is more closely associated with clay minerals, further decay appears more slowly and gradually. Soils and sediments thus "mature" over very long periods and gradually acquire properties such as those described in the present invention, which instinctively attracts mammals to geophagy.

[0028] Iron in sand and clay, in its trivalent oxidation state (III), binds very strongly to the phenol / quinone groups of humic polymers, forming black insoluble materials, such as those found in surface geological sediments like lignite and in black forest soils. The "newer" humic materials produced in the anaerobic parts of freshwater lakes are yellowish or light brown. Both the black oxidized humic substances in lignite sediments and the soluble yellowish / brown humic materials formed in anaerobic freshwater environments are chemical derivatives of the plant lignocellulose fibers that were once present in green plants. However, despite both being derivatives of the same precursor - the plant's lignocellulose structure - as shown in the following examples, they are significantly different in their GM regulatory ability.

[0029] Need for the product The composition of the product named "Deconstructed Soil" (DS) is a sterilized substitute for soil as a GM regulatory composition. It is based on natural soil components, and its use is for preventing or combating dysbiosis through the application of a new principle that is fundamentally different from the current product market aimed at GM regulation. The current range of products regarded as GM regulators are commonly classified into three categories.

[0030] (1) Probiotics - preparations containing live bacteria or other microorganisms, (2) Prebiotics - energy substrates that selectively support the growth of gut bacteria presumed to be beneficial, (3) Postbiotics - metabolites left after culturing microorganisms.

[0031] These product categories correspond to large markets, but they do not adequately consider the complex ecological environment of GM, which is a community composed of hundreds of species that form a highly complex metabolic interdependent network. For example, the introduction of a number of probiotic bacterial species (mostly lactic acid bacteria), whether one or a few, will inevitably change the ecological balance among species within the GM if the introduced bacteria survive and become new members of the GM. Prebiotic substances (mostly oligosaccharides) have been promoted as a means to advantageously promote the growth of a limited number of species (presumed to be beneficial) within the GM, and thus prebiotics may induce changes in the GM ecological environment. The adverse effects of probiotics sold as being good for human and animal health have been hypothesized based on theoretical knowledge of the GM ecological environment, but there are also significant warnings based on solid scientific tests. As an example, probiotics may delay the normal recovery of the human GM after antibiotic treatment, contrary to what is commonly believed and recommended medical practice. Also, prebiotics as defined above do not conform to the basic ecological principles of the GM, and their effectiveness in promoting gut health is doubtful.

[0032] Therefore, there is an unmet need for products that regulate GM according to the basic laws of the ecological environment of microorganisms in anaerobic environments, such as the DS of the present invention. Mechanism of action The black humic material component of DS is a polymer of aromatic monomers containing phenolic and quinone moieties that chelate iron(III). This structure can serve as an electron shuttling agent in anaerobic environments. In this process, iron(III) can be reduced to iron(II), and the quinone moiety can be reduced to semiquinone and phenolic groups. The reduced iron(II) and phenolic moieties in the polymer of the humic material can, in turn, deliver electrons to other microorganisms within the ecosystem and be re-oxidized. This provides a means by which fermentative microorganisms can reduce excess reducing equivalents, thereby redistributing energy to other community members. From our current understanding of anaerobic metabolism, this mechanism of action is insufficient to explain all of our observations, such as the fact that there are both obligate anaerobic species that are favored by DS and obligate anaerobic species that are not favored. For example, a species of the genus Bacteroides, which is within the obligate anaerobic group, was among the most widely observed groups in our control experiments, but with treatment using DS, these species were almost removed from the assay cultures. This is clearly in contrast to the action of a purified humic material derived from fresh water that showed no action on these species compared to the control. In addition, the growth of Clostridium perfringens, which is presumed to be harmful, was enhanced by the lignin component, but DS eliminated this species. Furthermore, lignin had no effect on the growth of Prevotella copri and Faecalibacterium prausnitzii, but DS significantly enhanced the same species. Therefore, more fundamental research is needed to clarify the detailed mechanism behind the discovery of the very unexpected GM-regulating properties of DS.

[0033] Potential uses The product of the present invention should be a basic formulation that can be used alone or in combination with other products in addressing conditions such as maintaining ecologically balanced gut microbiota and conditions related to gut dysbiosis, obesity, alcoholic and non-alcoholic fatty liver diseases, autism, type II diabetes, cardiovascular health, inflammatory bowel disease, irritable bowel syndrome, metabolic syndrome, myalgic encephalomyelitis / chronic fatigue syndrome, ADHD, Parkinson's disease, AIDS, depression, arthritis, allergies, etc. It can also be used as a basic prescription to assist in fecal microbiota transplantation (FMT) in preventing the abnormal growth of Clostridium difficile. It can also be used as a microbiome-modulating adjuvant for cancer drugs and cancer vaccines, including cancer immunotherapy, and for general vaccines. For example, as shown in the examples, this can be achieved by selectively enhancing the growth of the genus Faecalibacterium and disadvantaging the growth of the genus Bacteroides.

[0034] The microbiota-regulating ability of DS is also implied in the discoveries described in the following examples to be valid for other uses beyond the prevention or treatment of gut dysbiosis, such as the regulation of microbiota in inflamed periodontal tissues causing wounds and inflammation.

[0035] It is also implied that DS may have uses in the commercial agriculture of birds (chickens, turkeys) and fish. Commercial chicken production from fertilized eggs to slaughter is carried out under a technical regime designed to avoid and prevent the exposure to microbiota to which chickens have adapted during evolution. To make chickens in such an environment more robust against accidental exposure to opportunistic pathogens in chicken production facilities, it could be an excellent strategy to beneficially enrich the anaerobic microbiota in their closed blind-sacks using a product such as DS.

[0036] Within the aquaculture sector, the modern farming method of tilapia is particularly suitable for the use of DS. This tropical fish species is biologically adapted to freshwater ecosystems rich in algae and microorganisms and with anaerobic bottom sludge. The fish regularly ingests the anaerobic bottom sludge and returns to the oxygen-rich water surface to eat phytoplankton. In modern farming methods in non-polluted pond water or in open net cages, this fish is deprived of such anaerobic input, which can be compared to geophagy in mammals. Therefore, the DS composition of the present invention may be an excellent substitute.

[0037] The deconstructed soil composition according to the present invention contains a black humic material containing β-1,3 / 1,6-glucan as well as minerals and polysaccharides. The β-1,3 / 1,6-glucan added to the black humic material is of the fungal type and is preferably obtained from yeast. Furthermore, the black humic material of the present composition contains iron and oxidized lignite / humic material. Preferably, the black humic material is of the Leonardite type. Optionally, the composition of the present invention contains clay minerals.

[0038] In a further embodiment of the present invention, the ratio of β-1,3 / 1,6-glucan to the black humic material ranges from 1:100 to 1:1, preferably 5:100, and most preferably 3.5:100.

[0039] When the deconstructed soil composition of the present invention contains clay minerals, the ratio of β-1,3 / 1,6-glucan to the black humic material to the clay minerals ranges from 1:100:5 to 5:100:15, preferably 3.5:100:10.

[0040] The use of the deconstructed soil composition as defined above in the treatment of dysbiosis in vertebrates, particularly mammals, is included within another embodiment of the present invention. According to another embodiment of the present invention, the mammal is a human, a pet or a livestock. Still further embodiments of the present invention include the use of the above composition where the vertebrate is a bird or an aquaculture species.

[0041] In yet another embodiment of the present invention, a deconstructed soil composition is used to selectively advantageously condition oxygen-sensitive beneficial gut bacteria under anaerobic and microaerophilic conditions. In further embodiments, a deconstructed soil composition is used to selectively disadvantageously condition oxygen consumption and harmful gut bacteria.

[0042] In certain embodiments of the present invention, a deconstructed soil composition is used to enhance the growth of Faecalibacterium prausnitzii, Prevotella copri, Akkermansia muciniphila, Methanobrevibacter smithii, species of the genus Bifidobacterium, and species of the genus Lactobacillus under anaerobic and / or microaerophilic conditions.

[0043] In other certain embodiments of the present invention, a deconstructed soil composition is used to disadvantageously condition the growth of Clostridium perfringens, Finegoldia magna, Alistipes shahii, the genus Staphylococcus, Bacter Roy des umiformis) and / or Bacter oi des vulgatus) under anaerobic and / or microaerophilic conditions. Roy des vulgatus) under anaerobic and / or microaerophilic conditions. oi des vulgatus) under anaerobic and / or microaerophilic conditions.

[0044] Products, Methods, and Experiments Test Components The deconstructed soil of the present invention contains the basic components present in forest soil, namely, 1) fungal type β-1,3 / 1,6-glucan, and 2) black humic material derived from geological sediments containing iron and residual polysaccharides, and 3) clay material. For comparison, 4) pure humic material derived from a freshwater lake, and 5) the GM regulatory properties of lignin derived from rye bran were tested.

[0045] 1) β-1,3 / 1,6-glucan To produce a representative type of soil fungal cell wall β-1,3 / 1,6-glucan, Saccharomyces cerevisiae was selected as the raw material, but filamentous fungi can also be used as a source of β-1,3 / 1,6-glucan.

[0046] The extraction procedure is as follows: A paste of live Saccharomyces cerevisiae cells was suspended in distilled water (50 grams / liter), and this suspension was stirred at 60 °C for 24 hours to autolyze the cells. Then, the insoluble matter mainly containing the crude cell wall was collected by centrifugation. To remove the soluble matter from the insoluble crude cell wall sediment, resuspension of the insoluble crude cell wall sediment in water and sedimentation by centrifugation were repeated. This washed cell wall preparation was suspended in sodium hydroxide at 60 - 70 °C for 5 hours to separate and partially decompose proteins and lipids from the cell wall polysaccharides, and a cycle of centrifugation and washing in water was repeated for the alkali-treated cell wall preparation to remove the alkali-soluble matter. The pH of the slurry of the alkali-treated and washed cell wall was adjusted to 7 using citric acid, and the neutralized slurry was heated to 80 °C and sieved through a 0.5 mm mesh filter cloth. The β-glucan content of the obtained cell wall paste was 65%, and the remaining part was mainly lipids. Preparations containing more than 80% β-glucan were made by extraction with ethanol.

[0047] This extraction procedure is the same as that used in the commercial production of particulate yeast β-1,3 / 1,6-glucan products such as NBG (Norwegian Beta Glucan) by Biotec Pharmacon ASA or WCBG by Biothera Inc., a US-based company.

[0048] As suitable carriers for the various humic material preparations in the deconstructed soil compositions of the present invention, all ethanol extraction preparations containing 60 wt%, 80 wt%, and 98 wt% of β-1,3 / 1,6-glucan were tested. The carrier properties of different particulate β-1,3 / 1,6-glucan preparations were considered excellent if they swelled and formed hydrogels when mixed with humic material preparations and clay and even when autoclaved (120°C / 20 min). Even when dry black humus or lignin was mixed before autoclaving, all β-1,3 / 1,6-glucan preparations tested (60%, 80%, and 98% β-glucan) had a swelling ability corresponding to at least 100 mL of aqueous gel when 5 grams of dry β-1,3 / 1,6-glucan was autoclaved in water.

[0049] Examples 1 to 30 show the results when using the commercially available β-1,3 / 1,6-glucan preparation M-Gard (Biotec Pharmacon ASA; www.biotec.no) containing 80% β-1,3 / 1,3-glucan. When this product was suspended in 5 grams (dry) of distilled water and autoclaved, it formed a gel volume of 100 ml, and some additional water was found in an independent layer. The same gel volume result was obtained when M-Gard was mixed with about 5 grams of dry black humic material, so it is an ideal carrier that can fully incorporate black humic material.

[0050] It should be noted that the amount of β-1,3 / 1,6-glucan and black humic material can be mixed in equal amounts without changing the general pattern of GM regulatory ability. 2) Black humic material Another main component of the deconstructed soil of the present invention was an unprocessed, iron-containing, highly oxidized (black) lignite / humic material preparation formed in the geological sediment (lignite) of the chemical and microbiological decay of organic matter that had continued for thousands of years. We recognized that the product was a representative of microbiologically and chemically modified plant lignin present in black forest soil and differed from native lignin and polysaccharides present in green leaves in chemical and other properties. The black humic material used in the experiments shown in Examples 1 to 30 was a water-insoluble black powder extracted from a Leonardite-type lignite produced in Münster, Germany, with an alkali. The powder had an ash content of 16% (after combustion at 480 °C) consisting mainly of iron(III) oxide.

[0051] We compared the microbiome-regulating properties of the DS of the present invention with those of native lignin extracted from rye bran and a pure yellowish (reduced, iron-free) humic material formed in a freshwater lake. We did this to examine whether the effects described in the examples could be attributed to a certain degree of "aging" and "maturing" of the humus between microbiological and chemical decay. The results from such comparative tests show that the action of the black humic material is significantly different from that of the pure humic material derived from fresh water (Examples 25 to 27) and native lignin derived from rye bran (Examples 28 to 30).

[0052] 3) Clay material The inorganic component added to the basic DS containing β-1,3 / 1,6-glucan and humic material was of the illite type available in health food stores. It contained aluminum silicate as the main component.

[0053] 4) Pure humic acid The pure humic acid used in the experiments presented in Examples 25 to 27 was brought about in relation to a research project in limnology at the University of Oslo. First, the yellowish clear water from an inland lake in the Oslo area was filtered to remove particulate matter (>1 micron), and then ultrafiltration (>10,000 D) was performed to concentrate the humic substances. After repeating the cycles of washing in water and ultrafiltration, the pure humic acid was vacuum dried to obtain a fluffy light brown powder with a microscopic microstructure of microfibers. This substance was used in the experiments presented in Examples 25 to 27.

[0054] 5) Lignin The lignin used in the experiments shown in Examples 28 to 30 was extracted from rye bran as follows: The dry rye bran (Norgesmollene AS, Norway) was finely ground to a particle size of 1 - 2 mm and suspended in water (1 weight unit / 50 mL), acidified to pH 1 - 2 using HCl, and 0.5 grams of porcine pepsin (Arctic Zymes AS, Norway) was added thereto. After pepsin digestion of this mixture at 37°C for 24 hours, the solid phase was sedimented and washed with water by repeating centrifugation and sedimentation. The white top layer (cellulose) in the sediment was aspirated, and the bottom layer containing brown lignin was repeatedly washed with 96% ethanol to remove any low molecular weight substances soluble in ethanol. The brown powder obtained after air drying was used in the experiments of Examples 28 to 30.

[0055] GM analysis technique It is only relatively recently that scientists have been able to describe GM in detail at all levels. The development of DNA-sequencing technology has led to an “explosion” of research into the human GM as well as other complex microbial ecosystems. These efforts have demonstrated the previously unknown complexity and individual nature of the microbial communities that inhabit the gut. The most common approach for investigating GM is known as marker gene or amplicon DNA sequencing. In the case of bacteria, this approach involves the next-generation sequencing of phylogenetic marker genes, which typically encode the small subunit RNA component (16S rRNA) of the ribosome, the cell's protein synthesis machinery. This gene is found in all bacteria and archaea and contains highly conserved regions as well as variable stretches, which makes it suitable for phylogenetic comparisons. The current gold standard in high-throughput DNA sequencing is the Illumina platform, which has the best cost-to-output ratio. There are several different technical approaches for preparing samples for amplicon sequencing on an Illumina device, the so-called DNA sequencing library preparation methods. One thing common to all of them is the amplification of target DNA fragments by PCR (polymerase chain reaction) technology. The details of this process as well as the downstream processing of the amplified DNA fragments vary substantially depending on the protocol used, as do the costs associated with library preparation and the quality of the output data.

[0056] We developed a novel methodology for 16S rRNA gene amplicon sequencing, including a downstream bioinformatics pipeline, to successfully characterize the microbiota on a large scale (thousands of samples) (de Muinck et al., 2017). Specifically, we developed a novel library preparation technique that incorporates a third index sequence as part of the second-step PCR amplification, reducing the number of oligonucleotides required for sequencing. The main idea was to add dual indices and some Illumina adapters during the first-step PCR, while in the second reaction, one common oligonucleotide was used for all reactions and one custom oligonucleotide was used for every 96 samples (one standard reagent plate), completing the adapter sequences so that only successfully amplified fragments were sequenced. We performed extensive benchmark testing and optimization of the technology. Since our technology is very flexible, we can use it to characterize complex microbial communities derived from various different sample types. Thereby, our method represents a significant advancement in amplicon sequencing of bacterial communities from a cost-to-output perspective and is ideally suited for processing large numbers of samples to produce the highest quality data.

[0057] Microbiome assay The above techniques were used to generate the data shown in the following examples regarding how the deconstructed soil (DS) of the present invention, consisting of β-1,3 / 1,6-glucan and black humic material, modulates a model of the human GM microbiota based on fecal material cultured either under strict anaerobic conditions or microaerophilic conditions. We also tested the effect of DS supplemented with the clay mineral illite. Cultures were grown for 7 days at 37 °C in 2 mL sterile tubes containing Anaerobe Basal broth (Oxoid), either as a control or with the addition of DS. For anaerobic conditions, cultures were grown in airtight bottles containing an anaerobic GasPak (Thermo) pouch and an indicator strip to confirm anaerobiosis. Microaerophilic cultures were grown in a standard incubator, covered with a loose lid on the tubes for the first 24 h. After 24 h, the lids were tightened. After 7 days, all cultures were frozen at -80 °C and awaited further processing. Total DNA was extracted from the cultures using the MagAttract PowerSoil DNA kit. The DNA was analyzed using the above sequencing procedure. Sequencing was performed until an average depth of 67,498 (±17,052 s.d.) reads was achieved. Sequence reads were classified to the genus level using the Ribosomal Database Project training set (Cole et al., 2014). Further classification to the species level was performed by BLAST search (Altschul et al., 1990) against the Genbank 16S rRNA gene sequence archive. If we found a high identity match (>99%) to a single species, this was presented as a classification of sequence variant. If the sequence was found to have 100% identity with more than one species, the sequence variant was presented at the genus level of the classification.

[0058] Summary of Examples Examples 1 - 6 demonstrate that under anaerobic conditions, deconstructed soil (DS) and deconstructed soil with illite added (DS+) have a beneficial effect on bacteria associated with good gut health in a complex gut microbiota culture. Furthermore, rare bacterial types are recovered.

[0059] Examples 7 - 12 demonstrate that under anaerobic and microaerophilic conditions, DS with illite added (DS+) has a beneficial effect on bacteria associated with good gut health in a complex gut microbiota culture. Furthermore, rare bacterial types are recovered.

[0060] Examples 13 - 15 demonstrate a specific property of the present invention, namely that multiple types of bacteria, which are also anaerobic, are not advantaged by the present invention. The examples presented are types that have been associated with gut health in a negative direction.

[0061] Example 16 demonstrates a specific property of the present invention. The facultative anaerobic bacterium Staphylococcus is not advantaged by the present invention. Staphylococcus is a member of the normal but minor GM community. An increase in the level of Staphylococcus can be an indicator of aerobic life in the GM.

[0062] Examples 17 - 18 demonstrate a specific property of the present invention, namely that bacterial types, which are also anaerobic but not necessarily associated with gut health either positively or negatively, are not advantaged by the present invention.

[0063] Examples 19 - 21 demonstrate a specific property of the present invention, namely that multiple types of bacteria, which are also anaerobic, are not advantaged by the present invention under microaerophilic conditions. The examples presented are types that have been associated with gut health in a negative direction.

[0064] Example 22 demonstrates certain properties of the present invention. The genus Staphylococcus, a facultative anaerobic bacterium, is not advantaged by the present invention under microaerophilic conditions. The genus Staphylococcus is a member of the normal but minor GM community. An increase in the level of the genus Staphylococcus can be an indicator of aerobic life in GM.

[0065] Examples 23 - 24 demonstrate certain properties of the present invention, namely that bacterial types which are similarly anaerobic but not necessarily related as good or bad for intestinal health are not advantaged by the present invention under microaerophilic conditions.

[0066] Examples 25 - 27 demonstrate the effect on GM of pure fresh - water humic material under anaerobic conditions, which shows that the effect of this humic material is significantly different from that of DS.

[0067] Examples 28 - 30 demonstrate the effect on GM of cereal - derived native lignin, calamus lignin, under anaerobic conditions, which shows that the effect of the native lignin is significantly different from that of DS.

Example

[0068] (Example 1) Increase in the relative abundance of the genus Bifidobacterium in an anaerobic human GM microbial assay system treated with deconstructed soil (DS, n = 5) and DS supplemented with illite (DS +, n = 4), compared to untreated control (C, n = 7).

[0069] The increase in abundance under both treatments is statistically significant compared to the control (p < 0.003, Wilcoxon rank - sum test). The horizontal line represents the mean value and the dots represent the actual data points.

[0070] The results are shown in Figure 1. Comment: Multiple beneficial effects on the human host are thought to be due to a species of the genus Bifidobacterium that is indigenous to the human intestine. Furthermore, the abundance of the genus Bifidobacterium has been found to be reduced in patients suffering from IBD, obesity, allergy, and autism.

[0071] (Example 2) Increase in the relative abundance of the genus Lactobacillus in an anaerobic human GM microbiota assay system treated with deconstructed soil (DS, n = 5) and DS supplemented with illite (DS+, n = 4), compared to untreated control (C, n = 7).

[0072] The increase in abundance under both treatments is statistically significant compared to the control (p < 0.01, Wilcoxon rank-sum test). The horizontal line represents the mean value, and the dots represent the actual data points.

[0073] The results are shown in Figure 2. Comment: A species of the genus Lactobacillus, such as L. rhamnosus or L. casei, is widely used in traditional food preservation techniques, and it is widely assumed that these foods have beneficial effects. A species of the genus Lactobacillus is also the most widely used probiotic bacterial group.

[0074] (Example 3) Increase in the relative abundance of Prevotella copri in an anaerobic human GM microbiota assay system treated with deconstructed soil (DS, n = 5) and DS supplemented with illite (DS+, n = 4), compared to untreated control (C, n = 7).

[0075] The increase in abundance under both treatments is statistically significant compared to the control (p < 0.01, Wilcoxon rank-sum test). The horizontal line represents the mean value, and the dots represent the actual data points.

[0076] The results are shown in Figure 3. Comment: Prevotella copri belongs to the phylum Bacteroidetes. They can be found quite widely in the human microbiome, especially in high abundances in communities of hunter-gatherers where Western lifestyle diseases do not actually exist. Furthermore, in patients with IBD, a reduced abundance of the genus Prevotella has been found compared to healthy controls.

[0077] (Example 4) Increase in the relative abundance of Faecalibacterium prausnitzii compared to untreated controls (C, n = 7) in an anaerobic human GM microbiota assay system treated with deconstructed soil (DS, n = 5) and DS supplemented with illite (DS+, n = 4).

[0078] The increase in abundance under both treatments is statistically significant compared to the control (p < 0.004, Wilcoxon rank-sum test). The horizontal line represents the mean value, and the dots represent the actual data points.

[0079] The results are shown in Figure 4. Comment: At present, Faecalibacterium prausnitzii is obligately anaerobic and contains only Faecalibacterium prausnitzii, which produces butyrate and is an anti-inflammatory species. This highly oxygen-sensitive, mucus-associated bacterium has been found to be drastically reduced in inflammatory bowel disease (IBD). Due to its extreme oxygen sensitivity, it is very difficult to grow this bacterium in culture and is thus a difficult bacterial candidate for development as a probiotic.

[0080] (Example 5) Increase in the relative abundance of Methanobrevibacter smithii compared to untreated controls (C, n = 7) in an anaerobic human GM microbiota assay system treated with deconstructed soil (DS, n = 5) and DS supplemented with illite (DS+, n = 4).

[0081] The increase in abundance under both treatments was statistically significant compared to the control (p < 0.02, Wilcoxon rank-sum test). The horizontal line represents the mean value, and the dots represent the actual data points.

[0082] The results are shown in Figure 5. Comment Methanobrevibacter smithii is an archaeal genus, and M. smithii has been associated with the human gut microbiota. It removes hydrogen gas, a common byproduct of bacterial metabolism, in the human intestine. This process is strictly anaerobic, and the M. smithii species is extremely sensitive to oxygen. A sharp decrease in M. smithii in the human intestine has been proposed as a biomarker for IBD.

[0083] (Example 6) Increase in relative abundance of Akkermansia muciniphila in an anaerobic human gut microbiota assay system treated with deconstructed soil (DS, n = 5) and DS supplemented with illite (DS+, n = 4), compared to untreated controls (C, n = 7).

[0084] The increase in abundance under both treatments was statistically significant compared to the control (p < 0.02, Wilcoxon rank-sum test). The horizontal line represents the mean value, and the dots represent the actual data points.

[0085] The results are shown in Figure 6. Comment Akkermansia muciniphila is a genus of obligate anaerobic bacteria and belongs to the phylum Verrucomicrobia. They have been shown to be drastically reduced in mouse models of type I diabetes and obesity. Akkermansia muciniphila bacteria are associated with metabolic health in humans and are recognized as excellent candidates for the next generation of probiotics.

[0086] (Example 7) Increase in the relative abundance of Bifidobacterium in a microaerophilic human GM microbiota assay system treated with deconstructed soil supplemented with illite (MDS+, n = 3), compared to an untreated control (MC, n = 5).

[0087] The corresponding anaerobic assay was included for comparison (AC = control, ADS+ = DS supplemented with illite). The increase in abundance in the cultures treated with MDS+ is statistically significant compared to the control (MC) (p < 0.05, Wilcoxon rank-sum test). The horizontal line represents the mean value, and the dots represent the actual data points.

[0088] The results are shown in Figure 7. (Example 8) Increase in the relative abundance of Lactobacillus in a microaerophilic human GM microbiota assay system treated with deconstructed soil supplemented with illite (MDS+, n = 3), compared to an untreated control (MC, n = 5).

[0089] The corresponding anaerobic assay was included for comparison (AC = control, ADS+ = DS supplemented with illite). The horizontal line represents the mean value, and the dots represent the actual data points. The results are shown in Figure 8.

[0090] (Example 9) Increase in the relative abundance of Prevotella copri in a microaerophilic human GM microbiota assay system treated with deconstructed soil supplemented with illite (MDS+, n = 3), compared to an untreated control (MC, n = 5).

[0091] The corresponding anaerobic assay was included for comparison (AC = control, ADS+ = DS supplemented with illite). The increase in abundance in the cultures treated with MDS+ is statistically significant compared to the control (MC) (p < 0.05, Wilcoxon rank-sum test). The horizontal line represents the mean value, and the dots represent the actual data points.

[0092] The results are shown in Figure 9. (Example 10) Increase in the relative abundance of Faecalibacterium prausnitzii in a microaerophilic human GM microbiota assay system treated with deconstructed soil supplemented with illite (MDS+, n = 3), compared to untreated controls (MC, n = 5).

[0093] The corresponding anaerobic assay was included for comparison (AC = control, ADS+ = DS supplemented with illite). The increase in abundance in the cultures treated with MDS+ was statistically significant compared to the control (MC) (p < 0.05, Wilcoxon rank-sum test). The horizontal line represents the mean value, and the dots represent the actual data points.

[0094] The results are shown in Figure 10. (Example 11) Increase in the relative abundance of Methanobrevibacter smithii in a microaerophilic human GM microbiota assay system treated with deconstructed soil supplemented with illite (MDS+, n = 3), compared to untreated controls (MC, n = 5).

[0095] The corresponding anaerobic assay was included for comparison (AC = control, ADS+ = DS supplemented with illite). The increase in abundance in the cultures treated with MDS+ was statistically significant compared to the control (MC) (p < 0.05, Wilcoxon rank-sum test). The horizontal line represents the mean value, and the dots represent the actual data points.

[0096] The results are shown in Figure 11. (Example 12) Increase in the relative abundance of Akkermansia muciniphila in a microaerophilic human GM microbiota assay system treated with deconstructed soil supplemented with illite (MDS+, n = 3), compared to untreated controls (MC, n = 5).

[0097] The corresponding anaerobic assay was included for comparison (AC = control, ADS+ = DS supplemented with illite). The increase in abundance in the cultures treated with MDS+ was statistically significant compared to the control (MC) (p < 0.05, Wilcoxon rank sum test). The horizontal line represents the mean value, and the dots represent the actual data points.

[0098] The results are shown in Figure 12. (Example 13) Decrease in the relative abundance of Clostridium perfringens compared to untreated control (C, n = 7) in an anaerobic human GM microbiota assay system treated with deconstructed soil (DS, n = 5) and DS supplemented with illite (DS+, n = 4).

[0099] The decrease in abundance under both treatments was statistically significant compared to the control (p < 0.003, Wilcoxon rank sum test). The horizontal line represents the mean value, and the dots represent the actual data points.

[0100] Comment Clostridium perfringens is widely associated with intestinal diseases in humans and animals. The results are shown in Figure 13.

[0101] (Example 14) Decrease in the relative abundance of Finegoldia magna compared to untreated control (C, n = 7) in an anaerobic human GM microbiota assay system treated with deconstructed soil (DS, n = 5) and DS supplemented with illite (DS+, n = 4).

[0102] The decrease in abundance under both treatments was statistically significant compared to the control (p < 0.05, Wilcoxon rank sum test). The horizontal line represents the mean value, and the dots represent the actual data points.

[0103] Comment Finegoldia magna acts as an opportunistic human pathogen. The results are shown in Figure 14. (Example 15) Decrease in relative abundance of Alistipes shahii compared to untreated control (C, n = 7) in an anaerobic human GM microbiota assay system treated with deconstructed soil (DS, n = 5) and DS supplemented with illite (DS+, n = 4).

[0104] The decrease in abundance under both treatments is statistically significant compared to the control (p < 0.01, Wilcoxon rank sum test). The horizontal line represents the mean value, and the dots represent the actual data points.

[0105] Comment An increased level of a species of the genus Alistipes has been associated with increased pain in children with irritable bowel syndrome. The results are shown in Figure 15.

[0106] (Example 16) Decrease in relative abundance of Staphylococcus compared to untreated control (C, n = 7) in an anaerobic human GM microbiota assay system treated with deconstructed soil (DS, n = 5) and DS supplemented with illite (DS+, n = 4). The horizontal line represents the mean value, and the dots represent the actual data points.

[0107] Comment The genus Staphylococcus is a facultative anaerobe and a member of the normal, but minor, GM community. An increase in the level of Staphylococcus can be an indicator of aerobic life in the GM.

[0108] The results are shown in Figure 16. (Example 17) Decrease in relative abundance of Bacteroides uniformis compared to untreated control (C, n = 7) in an anaerobic human GM microbiota assay system treated with deconstructed soil (DS, n = 5) and DS supplemented with illite (DS+, n = 4).

[0109] The decrease in abundance under both treatments is statistically significant compared to the control (p < 0.006, Wilcoxon rank-sum test). The horizontal line represents the mean value, and the dots represent the actual data points.

[0110] The results are shown in Figure 17. (Example 18) Decrease in relative abundance of Bacteroides vulgatus compared to untreated control (C, n = 7) in an anaerobic human GM microbiota assay system treated with deconstructed soil (DS, n = 5) and DS supplemented with illite (DS+, n = 4).

[0111] The decrease in abundance under both treatments is statistically significant compared to the control (p < 0.006, Wilcoxon rank-sum test). The horizontal line represents the mean value, and the dots represent the actual data points.

[0112] The results are shown in Figure 18. (Example 19) Decrease in relative abundance of Clostridium perfringens compared to untreated control (MC, n = 5) in a microaerophilic human GM microbiota assay system treated with deconstructed soil supplemented with illite (MDS+, n = 3).

[0113] The corresponding anaerobic assay was included for comparison (AC = control, ADS+ = DS supplemented with illite). The decrease in abundance in the cultures treated with MDS+ is statistically significant compared to the control (MC) (p < 0.05, Wilcoxon rank-sum test). The horizontal line represents the mean value, and the dots represent the actual data points.

[0114] The results are shown in Figure 19. (Example 20) Decrease in the relative abundance of Finegoldia magna in a microaerophilic human GM microbiota assay system treated with illite-supplemented deconstructed soil (MDS+, n = 3), compared to an untreated control (MC, n = 5).

[0115] The corresponding anaerobic assay was included for comparison (AC = control, ADS+ = DS supplemented with illite). The decrease in abundance in the cultures treated with MDS+ was statistically significant compared to the control (MC) (p < 0.05, Wilcoxon rank-sum test). The horizontal line represents the mean value, and the dots represent the actual data points.

[0116] The results are shown in Figure 20. (Example 21) Decrease in the relative abundance of Alistipes shahii in a microaerophilic human GM microbiota assay system treated with illite-supplemented deconstructed soil (MDS+, n = 3), compared to an untreated control (MC, n = 5).

[0117] The corresponding anaerobic assay was included for comparison (AC = control, ADS+ = DS supplemented with illite). The decrease in abundance in the cultures treated with MDS+ was statistically significant compared to the control (MC) (p < 0.04, Wilcoxon rank-sum test). The horizontal line represents the mean value, and the dots represent the actual data points.

[0118] The results are shown in Figure 21. (Example 22) Decrease in the relative abundance of Staphylococcus in a microaerophilic human GM microbiota assay system treated with illite-supplemented deconstructed soil (MDS+, n = 3), compared to an untreated control (MC, n = 5).

[0119] The corresponding anaerobic assay was included for comparison (AC = control, ADS+ = DS supplemented with illite). The horizontal line represents the mean value, and the dots represent the actual data points. The results are shown in Figure 22.

[0120] (Example 23) Decrease in relative abundance of Bacteroides uniformis in a microaerophilic human GM microbiota assay system treated with deconstructed soil supplemented with illite (MDS+, n = 3), compared to untreated control (MC, n = 5).

[0121] The corresponding anaerobic assay was included for comparison (AC = control, ADS+ = DS supplemented with illite). The decrease in abundance in the cultures treated with MDS+ was statistically significant compared to the control (MC) (p < 0.04, Wilcoxon rank sum test). The horizontal line represents the mean value, and the dots represent the actual data points.

[0122] The results are shown in Figure 23. (Example 24) Decrease in relative abundance of Bacteroides vulgatus in a microaerophilic human GM microbiota assay system treated with deconstructed soil supplemented with illite (MDS+, n = 3), compared to untreated control (MC, n = 5).

[0123] The corresponding anaerobic assay was included for comparison (AC = control, ADS+ = DS supplemented with illite). The decrease in abundance in the cultures treated with MDS+ was statistically significant compared to the control (MC) (p < 0.04, Wilcoxon rank sum test). The horizontal line represents the mean value, and the dots represent the actual data points.

[0124] The results are shown in Figure 24. (Example 25) Increase in relative abundance of the genus Bifidobacterium in an anaerobic human GM microbiota assay system treated with deconstructed soil (DS, n = 5) and DS supplemented with illite (DS+, n = 4), compared to untreated control (C, n = 7) and pure humic material derived from fresh water (PHA, n = 4).

[0125] The average relative abundance under PHA treatment is significant compared to the control (p = 0.04, Wilcoxon rank-sum test), but the effect of PHA is clearly reduced compared to DS and DS+. The horizontal line represents the mean value, and the dots represent the actual data points.

[0126] The results are shown in Figure 25. (Example 26) Decrease in relative abundance of Bacteroides uniformis in an anaerobic human GM microbiota assay system treated with deconstructed soil (DS, n = 5) and DS supplemented with illite (DS+, n = 4), compared to untreated control (C, n = 7) and pure humic material derived from fresh water (PHA, n = 4).

[0127] Unlike DS and DS+, PHA treatment is not significantly different from the control. The horizontal line represents the mean value, and the dots represent the actual data points. The results are shown in Figure 26.

[0128] (Example 27) Decrease in relative abundance of Bacteroides vulgatus in an anaerobic human GM microbiota assay system treated with deconstructed soil (DS, n = 5) and DS supplemented with illite (DS+, n = 4), compared to untreated control (C, n = 7) and pure humic material derived from fresh water (PHA, n = 4).

[0129] In PHA treatment, the relative abundance is significantly increased compared to the control (p = 0.02, Wilcoxon rank-sum test), while DS and DS+ had the opposite effect. The horizontal line represents the mean value, and the dots represent the actual data points.

[0130] The results are shown in Figure 27. (Example 28) Decreased relative abundance of Clostridium perfringens in an anaerobic human GM microbiota assay system treated with deconstructed soil (DS, n = 5) and DS supplemented with illite (DS+, n = 4), compared to untreated control (C, n = 7) and lignin (Lig, n = 5).

[0131] In the lignin treatment, the relative abundance increased significantly compared to the control (p < 0.01, Wilcoxon rank-sum test), while DS and DS+ had the opposite effect. The horizontal line represents the mean value, and the dots represent the actual data points.

[0132] The results are shown in Figure 28. (Example 29) Increased relative abundance of Prevotella copri in an anaerobic human GM microbiota assay system treated with deconstructed soil (DS, n = 5) and DS supplemented with illite (DS+, n = 4), compared to untreated control (C, n = 7) and lignin (Lig, n = 5).

[0133] The lignin treatment was not significantly different from the control, while the relative abundance increased with DS and DS+ treatments. The horizontal line represents the mean value, and the dots represent the actual data points. The results are shown in Figure 29.

[0134] (Example 30) Increased relative abundance of Faecalibacterium prausnitzii in an anaerobic human GM microbiota assay system treated with deconstructed soil (DS, n = 5) and DS supplemented with illite (DS+, n = 4), compared to untreated control (C, n = 7) and lignin (Lig, n = 5).

[0135] Although there was no significant difference from the control in the lignin treatment, the relative abundance increased in the DS and DS+ treatments. The horizontal line represents the average value, and the dots represent the actual data points. The results are shown in Fig. 30.

[0136] (References)

[0137] [Table 1]

Claims

A deconstructed soil composition for use in the prevention or treatment of intestinal microbiota dysbiosis in a target organism selected from the group consisting of mammals, birds, and aquaculture species, comprising a black humic material and β-glucan, wherein said β-glucan is a fungal type β-1,3 / 1,6-glucan that is resistant to microbial degradation, and said black humic material comprises iron and lignocellulose, the composition.

2. The composition according to claim 1, wherein said β-glucan is a β-1,3 / 1,6-glucan obtained from yeast.

3. The composition according to claim 1 or 2, wherein said black humic material is of the Leonardite type.

4. The composition according to any one of claims 1 to 3, comprising a clay mineral.

5. The composition according to any one of claims 1 to 4, wherein the ratio of β-1,3 / 1,6-glucan to black humic material is in the range of 1:100 to 1:

1.

6. The composition according to claim 5, wherein the ratio of β-1,3 / 1,6-glucan to black humic material is 5:100 or 3.5:

100.

7. The composition according to claim 4, wherein the ratio of β-1,3 / 1,6-glucan to black humic material to clay mineral is in the range of 1:100:5 to 5:100:

15.

8. The composition according to claim 7, wherein the ratio of β-1,3 / 1,6-glucan to black humic material to clay mineral is 3.5:100:

10.

9. The composition according to claim 1, wherein said black humic material is not pure humic acid.

10. The composition according to claim 1, not containing live microorganisms.

11. The composition according to any one of claims 1 to 10, wherein the mammal is a human.

12. The composition according to any one of claims 1 to 10, wherein the mammal is a pet or a livestock.

13. The composition according to any one of claims 1 to 10, wherein beneficial oxygen-sensitive intestinal bacteria are selectively favored under anaerobic and microaerophilic conditions.

14. The composition according to any one of claims 1 to 10, wherein harmful oxygen-sensitive intestinal bacteria are selectively disadvantaged under anaerobic and microaerophilic conditions.

15. The composition according to any one of claims 1 to 10, which enhances the growth of Faecalibacterium prausnitzii, Prevotella copri, Akkermansia muciniphila, Methanobrevibacter smithii, Bifidobacterium, and / or Lactobacillus under anaerobic and microaerophilic conditions.

16. The composition according to any one of claims 1 to 10, wherein the growth of Clostridium perfringens, Finegoldia magna, Alistipes shahii, Staphylococcus, Bacteroides uniformis, and / or Bacteroides vulgatus is disadvantaged under anaerobic and microaerophilic conditions.

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