How to use intestinal bacteria

Abstract

To provide a technique of acquiring and using useful bacterium included in feces of an individual.SOLUTION: Feces collection liquid containing feces collected from a human is cultured on M2GSC culture medium, genus Lactobacillus is isolated as useful bacteria derived from an enterobacterial flora of the human, the useful bacteria are cultured, the cultured useful bacteria are preserved, a substance using the useful bacteria including at least any one of the preserved useful bacteria, or metabolite or an extract thereof is obtained, and the substance using the useful bacteria is provided for the human individual who is the same person whom the feces was collected.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to techniques that can be used to realize a gut bacteria bank. [Background technology]

[0002] Food ingested by humans is digested by digestive juices secreted by the digestive tract, but it is known that a vast number of microorganisms present in the intestine assist in food digestion. These microorganisms, also known as the intestinal flora, not only aid in food digestion but also play roles in activating the immune system, metabolism, vitamin synthesis, and regulating the nervous system. In recent years, it has also become clear that the intestinal flora is associated with various diseases, including obesity, allergies, Parkinson's disease, depression, and cancer. Therefore, the intestinal flora, or the intestinal microbiome, a community of microorganisms, has been attracting attention for its important role in maintaining human health.

[0003] For example, Japanese Patent Application Laid-Open No. 2021-112218 (Patent Document 1) describes an invention relating to a composition that adjusts the composition of the intestinal flora, stating that changes in the composition of the intestinal flora are the cause of diseases, etc. Furthermore, for example, Japanese Patent Application Laid-Open No. 2021-109865 (Patent Document 2) describes an invention relating to an agent for improving the intestinal flora.

[0004] Meanwhile, techniques for collecting and preserving microorganisms contained in human feces are also known, and U.S. Patent Application Publication No. 2022 / 0160337 (Patent Document 3) describes a kit and method for collecting feces. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication No. 2021-112218 [Patent Document 2] Patent Publication No. 2021-109865 [Patent Document 3] US Patent Application Publication No. 2022 / 0160337 Summary of the Invention [Problem to be solved by the invention]

[0006] Although the group of bacteria is simply called the intestinal flora, the types and amounts of microorganisms present in the intestinal flora vary from person to person, and it is known that the types and amounts of so-called good bacteria that are considered to be beneficial to the body and bad bacteria that are considered to be harmful to the body also vary from person to person. However, the prior art described in Patent Document 1 or Patent Document 2 above only focuses on the general intestinal flora, and does not focus on the intestinal flora that differs from person to person. Therefore, it lacks a perspective of trying to improve the intestinal flora in a customized manner according to the characteristics of each individual.

[0007] Furthermore, although the conventional technology described in Patent Document 3 above allows for the collection of microorganisms from human feces, there is no knowledge whatsoever regarding the use of the collected microorganisms, and no technology is disclosed that aims to improve intestinal bacteria in a custom-made manner according to individual characteristics.

[0008] The applicant of this patent focuses on protecting or activating the beneficial bacteria in an individual's intestinal flora with the aim of creating a favorable intestinal environment for humans, and provides a gut bacteria bank business as an initiative to achieve this. The gut bacteria bank business is one of the services that focuses on an individual's microbiome, and involves analyzing an individual's intestinal flora, collecting, cultivating, and storing the beneficial bacteria contained in the intestinal flora, and providing the beneficial bacteria or supplements that utilize them.

[0009] The present disclosure provides techniques that contribute to the conservation and utilization of intestinal bacteria. [Means for solving the problem]

[0010] A first aspect of the present disclosure is a method for utilizing intestinal bacteria that utilizes useful bacteria in intestinal bacteria, comprising a bacterial flora analysis step of performing bacterial flora analysis on feces collected from a human; an isolation step of mixing the feces collected from the human into a feces collection kit containing a predetermined bacterial preservative solution and a stirring ball, and isolating useful bacteria from the feces collection solution after a predetermined period of time has passed; a culture step of culturing the isolated useful bacteria; a selection step of selecting excellent useful bacteria from the useful bacteria obtained in the culture step; a storage step of storing the selected useful bacteria; and a method for utilizing the stored useful bacteria, or metabolic products or extracts of the useful bacteria. and a useful bacteria utilization substance production step for obtaining a useful bacteria utilization substance containing at least one of the above. The isolation step includes a useful bacteria isolation step of culturing the collected stool fluid in any one of M2GSC medium, MRS medium, or MRS medium containing bile acid (cholic acid) to isolate Lactobacillus, and a useful bacteria isolation step of culturing the collected stool fluid in any one of MGLP agar medium, modified BCP-supplemented plate count agar medium, TOS mupirocin medium, TOS propionic acid agar medium, or paromomycin-containing TOS propionic acid agar medium to isolate Bifidobacterium.

[0011] A first aspect of the present disclosure includes a bacterial flora analysis step of analyzing the flora of feces collected from a human, thereby making it possible to obtain information about the types of bacteria that the human has in his or her body through the bacterial flora analysis; an isolation step of mixing the feces collected from the human into a feces collection kit containing a predetermined bacterial preservative solution and a stirring ball, and isolating useful bacteria from the feces collection solution after a predetermined period of time has passed, thereby making it possible to isolate and obtain useful bacteria that the human has; a culture step of culturing the isolated useful bacteria, thereby making it possible to increase the isolated useful bacteria; a selection step of selecting excellent useful bacteria from the useful bacteria obtained in the culture step, thereby making it possible to exert the effects of that type of bacteria at a high level; and a storage step of storing the selected useful bacteria, thereby making it possible to obtain the selected useful bacteria as desired. and a useful bacteria utilization substance production step for obtaining a useful bacteria utilization substance containing at least one of the stored useful bacteria or a metabolic product or extract of the useful bacteria, thereby making it possible to obtain a variety of useful products based on the useful bacteria. The isolation step includes a useful bacteria isolation step of culturing the fecal collection in any of M2GSC medium, MRS medium, or MRS medium containing bile acid (cholic acid) to isolate Lactobacillus, and culturing the fecal collection in any of MGLP agar medium, modified BCP-supplemented plate count agar medium, TOS mupirocin medium, TOS propionic acid agar medium, or paromomycin-containing TOS propionic acid agar medium to isolate Bifidobacterium, thereby enabling the preferred isolation of Lactobacillus and Bifidobacterium.

[0012] A second aspect of the present disclosure is a method for utilizing enteric bacteria that utilizes useful bacteria present in enteric bacteria, comprising an isolation step of mixing feces collected from a human with a predetermined bacterial preservative solution and isolating useful bacteria from the collected fecal solution after a predetermined period of time has passed, the isolation step comprising culturing the collected fecal solution in any of M2GSC medium, MRS medium, or MRS medium containing bile acid (cholic acid) to isolate Lactobacillus, and culturing the collected fecal solution in any of MGLP agar medium, modified BCP-supplemented plate count agar medium, TOS mupirocin medium, TOS propionic acid agar medium, or paromomycin-containing TOS propionic acid agar medium to isolate Bifidobacterium.

[0013] A second aspect of the present disclosure includes an isolation step of mixing feces collected from a human with a predetermined bacterial preservative solution and isolating useful bacteria from the fecal collection solution after a predetermined period of time has passed, thereby making it possible to isolate and obtain useful bacteria that the human possesses. The isolation step includes a step of culturing the fecal collection solution in any of M2GSC medium, MRS medium, or MRS medium containing bile acid (cholic acid) to isolate Lactobacillus, and a step of culturing the fecal collection solution in any of MGLP agar medium, modified BCP-supplemented plate count agar medium, TOS mupirocin medium, TOS propionic acid agar medium, or paromomycin-containing TOS propionic acid agar medium to isolate Bifidobacterium, thereby enabling the preferred isolation of Lactobacillus and Bifidobacterium.

[0014] A third aspect of the present disclosure is a method for utilizing intestinal bacteria that utilizes useful bacteria contained in intestinal bacteria, and includes an isolation step of mixing feces collected from a human with a predetermined bacterial preservative solution and isolating useful bacteria from the collected fecal solution after a predetermined period of time has passed, wherein the isolation step includes a step of culturing the collected fecal solution on any of BCP-added plate count agar medium, BCP plate medium, M2GSC medium, or calcium carbonate-containing MRS agar medium, and selecting colonies having the characteristics of desired useful bacteria from all the obtained colonies, thereby isolating the desired useful bacteria.

[0015] A third aspect of the present disclosure includes a step of culturing the collected stool fluid on any of BCP-added plate count agar medium, BCP plate medium, M2GSC medium, or calcium carbonate-containing MRS agar medium, and isolating the desired useful bacteria by selecting colonies having the characteristics of the desired useful bacteria from all the resulting colonies. Therefore, by using a medium in which multiple bacterial species can grow, the effort of culturing in multiple media can be eliminated.

[0016] A fourth aspect of the present disclosure is a method for using intestinal bacteria, further comprising a step of isolating at least one bacterium of the genus Faecalibacterium, Roseburia, Akkermansia, Collinsella, Pediococcus, or Blautia.

[0017] The fourth aspect of the present disclosure further includes a step of isolating at least one bacterium from the genus Faecalibacterium, Roseburia, Akkermansia, Collinsella, Pediococcus, or Blautia, and therefore useful bacteria other than Lactobacillus and Bifidobacterium can also be isolated and used.

[0018] A fifth aspect of the present disclosure is a method for isolating useful bacteria contained in feces collected from a human, which comprises mixing the feces with a bacterial preservative solution to obtain a fecal collection solution, culturing the resulting fecal collection solution in any of M2GSC medium, MRS medium, or MRS medium containing bile acid (cholic acid) to isolate Lactobacillus, and culturing the fecal collection solution in any of MGLP agar medium, modified BCP-supplemented plate count agar medium, TOS mupirocin medium, TOS propionic acid agar medium, or paromomycin-containing TOS propionic acid agar medium to isolate Bifidobacterium.

[0019] A fifth aspect of the present disclosure is a method for isolating useful bacteria contained in feces collected from a human, in which a fecal collection solution obtained by mixing the feces with a bacterial preservative solution is cultured in any of M2GSC medium, MRS medium, or MRS medium containing bile acid (cholic acid) to isolate Lactobacillus, and the fecal collection solution is cultured in any of MGLP agar medium, modified BCP-supplemented plate count agar medium, TOS mupirocin medium, TOS propionic acid agar medium, or paromomycin-containing TOS propionic acid agar medium to isolate Bifidobacterium, thereby enabling the preferred isolation of Lactobacillus and Bifidobacterium.

[0020] A sixth aspect of the present disclosure is a method for isolating useful bacteria contained in feces collected from a human, which comprises mixing the feces with a bacterial preservative solution to obtain a fecal collection solution, culturing the resulting solution on either BCP-added plate count agar medium, BCP plate medium, M2GSC medium, or calcium carbonate-containing MRS agar medium, and selecting colonies having the characteristics of the desired useful bacteria from all of the resulting colonies to isolate the desired useful bacteria.

[0021] A sixth aspect of the present disclosure is a method for isolating useful bacteria contained in feces collected from a human, in which the feces are mixed with a bacterial preservative solution to obtain a fecal collection solution, which is then cultured on either BCP-added plate count agar medium, BCP plate medium, M2GSC medium, or calcium carbonate-containing MRS agar medium, and the desired useful bacteria are isolated by selecting colonies having the characteristics of the desired useful bacteria from all the resulting colonies.Therefore, by using a medium in which multiple bacterial species can grow, the effort of culturing on multiple media can be avoided.

[0022] A seventh aspect of the present disclosure is a method for isolating useful bacteria, further comprising isolating at least one bacterium of the genus Faecalibacterium, Roseburia, Akkermansia, Collinsella, Pediococcus, or Blautia.

[0023] The seventh aspect of the present disclosure further isolates at least one bacterium from the genera Faecalibacterium, Roseburia, Akkermansia, Collinsella, Pediococcus, or Blautia, thereby making it possible to isolate useful bacteria other than Lactobacillus and Bifidobacterium.

[0024] Another aspect of the present disclosure is a method for obtaining useful bacteria, which comprises preparing a bacterial preservation solution in a buffer solution such as Dulbecco's phosphate-buffered saline, containing a reducing agent such as L-cysteine ​​hydrochloride and a cryoprotectant such as glycerol, and adjusting the pH to 3.5 to 5 with a pH adjuster such as sodium bicarbonate, mixing 1 to 2 g of feces into 5 to 10 mL of the bacterial preservation solution, and culturing the collected fecal solution after a predetermined period of time in a medium such as M2GSC medium to isolate useful bacteria such as Lactobacillus.

[0025] In another embodiment, a bacterial preservation solution is prepared by adding a reducing agent such as L-cysteine ​​hydrochloride and a cryoprotectant such as glycerol to a buffer solution such as Dulbecco's phosphate-buffered saline, adjusting the pH to 3.5 to 5 with a pH adjuster such as sodium bicarbonate, mixing 1 to 2 g of feces with 5 to 10 mL of the bacterial preservation solution, and culturing the collected fecal solution after a predetermined period of time in a medium such as M2GSC medium to isolate useful bacteria such as Lactobacillus. This method allows useful bacteria derived from an individual's intestinal flora to be obtained from the individual's feces. This can be useful in the future for maintaining and improving the intestinal flora.

[0026] Another embodiment of the present disclosure is a bacterial preservation solution containing L-cysteine ​​hydrochloride and glycerol in Dulbecco's phosphate buffered saline, the pH of which is adjusted to 3.5 to 5 with sodium bicarbonate.

[0027] The other embodiment is a bacterial preservative solution containing L-cysteine ​​hydrochloride and glycerol in Dulbecco's phosphate buffered saline, and the pH of which is adjusted to 3.5 to 5 with sodium bicarbonate, so that collected feces can be preserved and useful bacteria present in the feces can be protected until they can be cultured after a predetermined period of time has passed.

[0028] Another embodiment of the present disclosure is a bacterial preservation solution containing 0.005 to 0.02 g of a reducing agent and 5 to 10 mg of a pH adjuster per 10 mL in total of a buffer solution and a cryoprotectant.

[0029] The other embodiment is a bacterial preservative solution containing 0.005 to 0.02 g of a reducing agent and 5 to 10 mg of a pH adjuster per 10 mL of buffer solution and cryoprotectant in total, so that the pH can be maintained within a predetermined range even after mixing with feces, making it a bacterial preservative solution suitable for maintaining useful bacteria.

[0030] Another embodiment of the present disclosure is a bacterial preservation solution in which a buffer solution contains a reducing agent and a cryoprotectant, and the pH is adjusted to 3.5 to 5 with a pH adjuster.

[0031] The other embodiment is a bacterial preservative solution in which the buffer solution contains a reducing agent and a cryoprotectant and the pH is adjusted to 3.5 to 5 with a pH adjuster, so that the pH can be maintained within a predetermined range even after mixing with feces, making it a bacterial preservative solution suitable for maintaining useful bacteria.

[0032] Another aspect of the present disclosure is a stool collection kit comprising 5 to 10 mL of any of the above bacterial preservative solutions.

[0033] The other embodiment is a stool collection kit that includes 5 to 10 mL of any of the bacterial preservative solutions described above. Therefore, even if a desired amount of stool is collected and mixed with the bacterial preservative solution, the pH of the stool collection solution in the stool collection kit can be adjusted to a desired pH, and useful bacteria present in the stool can be protected until they can be cultured after a predetermined period of time has passed.

[0034] Another aspect of the present disclosure is a method for isolating useful bacteria, which comprises culturing collected fecal fluid in M2GSC medium to isolate Lactobacillus, and culturing it in paromomycin-containing TOS propionic acid agar medium to isolate Bifidobacterium.

[0035] The other embodiment is a method for isolating useful bacteria in which collected fecal fluid is cultured in M2GSC medium to isolate Lactobacillus, and then cultured in paromomycin-containing TOS propionic acid agar medium to isolate Bifidobacterium, so that useful bacteria, Lactobacillus and Bifidobacterium, derived from the individual who deposited the feces can be isolated from the feces.

[0036] Another aspect of the present disclosure is a method for isolating useful bacteria, such as Lactobacillus, by culturing collected fecal fluid in M2GSC medium or the like.

[0037] The other embodiment is a method for isolating useful bacteria in which collected stool fluid is cultured in M2GSC medium or the like to isolate useful bacteria such as Lactobacillus, and therefore necessary useful bacteria can be isolated from collected stool fluid.

[0038] Another aspect of the present disclosure is a method for utilizing intestinal bacteria obtained from a subject, the method comprising the steps of isolating useful bacteria from a bacterial preservative solution that has been contaminated with feces collected from the subject and allowed to stand for a predetermined period of time, freezing and preserving the isolated useful bacteria, culturing the frozen and preserved useful bacteria, and obtaining a substance containing the cultured useful bacteria or at least any of a metabolic product or extract of the cultured useful bacteria.

[0039] Another aspect of the present invention is a method for utilizing intestinal bacteria obtained from a subject, which includes the steps of isolating useful bacteria from a bacterial preservative solution that has been contaminated with feces collected from the subject and allowed to stand for a predetermined period of time, freezing and preserving the isolated useful bacteria, culturing the frozen and preserved useful bacteria, and obtaining an inclusion containing at least one of the cultured useful bacteria and a metabolic product or extract of the cultured useful bacteria. Therefore, the useful bacteria actually present in the body of the subject can be utilized. Then, an inclusion such as a capsule, supplement, or preparation containing a substance derived from the useful bacteria can be manufactured. [Effects of the Invention]

[0040] According to one aspect of the present disclosure, useful bacteria can be obtained from feces. According to another aspect of the present disclosure, bacteria contained in feces can be protected. According to yet another aspect of the present disclosure, feces suitable for obtaining beneficial bacteria can be secured and protected. According to yet another aspect of the present disclosure, useful bacteria can be isolated. According to yet another aspect of the present disclosure, the useful bacteria can be used to produce products, etc. [Brief explanation of the drawings]

[0041]

Figure 1

Figure 2

Figure 3

[0042] One aspect of the present disclosure will be described based on an exemplary embodiment with reference to the drawings. The following embodiment does not unduly limit the content of the present invention described in the claims. Furthermore, not all of the configurations described in the present embodiment are necessarily essential as a solution to the present invention.

[0043] All embodiments and optional embodiments included in this disclosure may be combined with each other to form new embodiments, and all technical features and optional technical features included in this disclosure may be combined with each other to form new technical features.

[0044] The term "or" used in this disclosure is used as an inclusive term. For example, "A or B" means "A, B, or both A and B." "A," "B," and "both A and B" all respectively satisfy "A or B."

[0045] In this specification and claims, when "first," "second," and "nth (where n is a natural number)" are used to distinguish between different elements, they are not intended to indicate a particular order, superiority, or inferiority. Configurations that are common to each embodiment are assigned the same reference numerals and redundant explanations will be omitted.

[0046] The directions or positional relationships indicated by terms such as "upper," "lower," "front," "rear," "left," and "right" and terms including these terms used in this specification and claims are based on the drawings and are merely for the purpose of conveniently and simply describing the embodiments. Therefore, unless expressly defined or limited, it is not intended to expressly or imply that a particular element or its use is configured in a particular direction, and it should be understood that this does not limit the scope of the claims and the embodiments.

[0047] Numerical values ​​or elements modified by the terms "about," "approximately," "nearly," or "substantially" as used in this specification and claims are understood to include the numerical value and any surrounding numerical values, and to include the element and anything that can be considered the same as the element. For example, when describing "about 3," "3" and any subsequent numerical values ​​can be included in "about 3" as long as the technical features and technical significance claimed in this disclosure are not different. Furthermore, when describing "B that is substantially identical to A," B can be completely identical to A, or even if there are differences, they can be included in the scope of "substantially" as long as the technical features and technical significance claimed in this disclosure are the same.

[0048] Unless otherwise specified, the symbol "~" used in this specification and claims is understood to include the lower limit and upper limit, as well as any value between the lower limit and upper limit. For example, "pH 3.5-5" means "pH 3.5 or higher and 5 or lower," and "0.005-0.02 g" means "0.005 g or higher and 0.02 g or lower."

[0049] One aspect of the present disclosure will be specifically described below.

[0050] 1. Bacterial flora analysis The human body is home to a diverse array of bacteria, known as resident flora. These bacteria vary widely across organs and tissues. For example, the skin is home to resident flora that adapt to its environment, while anaerobic bacteria exist in digestive organs like the intestines, where oxygen is difficult to reach. Traditional approaches to studying resident flora have focused on pathogenic bacteria, which cause illness and poor health, with the goal of eliminating or suppressing these bacteria. However, identifying only the pathogenic bacteria that harm the human body has not truly contributed to human health. For example, as we approach a 100-year lifespan, where it is too late to become ill once illness sets in, the goal has shifted from simply living a long, healthy life to simply living a long, healthy life. As the need for constant health and avoiding illness grows, simply studying and combating pathogenic bacteria is no longer sufficient.

[0051] In recent years, research in the field of genetics has progressed and analytical techniques have advanced. For example, the advent of next-generation sequencers has made it possible to analyze bacterial flora, which was previously difficult. This next-generation sequencer technology makes it possible to analyze bacteria that have not previously been identified because they are difficult to cultivate. From another perspective, it is now possible to analyze the bacterial flora, including not only bacteria that are harmful to the human body, such as pathogenic bacteria, but also bacteria that contribute to health promotion and bacteria that contribute to both (opportunistic bacteria). It has been found that visualizing the bacterial flora, including these various bacteria, is extremely useful for understanding the state of human health. Therefore, focusing on the fact that the state of the intestinal flora is closely related to maintaining and promoting human health, and that the intestinal flora can be analyzed using next-generation sequencers, we have developed the service described below with the aim of making the above knowledge more useful on an individual basis.

[0052] 2. Gut microbiota analysis service

[0053] Since the intestinal flora of humans is not the same for everyone but differs from person to person, it is necessary to first analyze the intestinal flora of a specific individual. For this purpose, it is preferable to collect an individual's feces and analyze the flora contained in that feces. We decided to analyze human feces to analyze the intestinal microbiota because both live and dead intestinal bacteria are excreted from the body in feces, making them easy to collect without using surgical procedures on the human body.

[0054] The use of next-generation sequencers is preferred for analyzing the intestinal microbiota. This is because next-generation sequencers can simultaneously determine the gene sequences of DNA or RNA samples from multiple bacterial species, allowing bacterial classification and identification based on a sequence database that stores bacterial gene sequences, their descriptions, and literature information. While the conventional Sanger sequencing method can only analyze a single gene sequence per bacterial species, next-generation sequencers are superior for analyzing the 16S rRNA gene of the intestinal microbiota, which contains a vast amount of genetic information.

[0055] Analysis using next-generation sequencers mainly involves classifying intestinal bacteria in feces by phylum, genus, and species. When classifying by phylum, genera and species are combined into one group, making it difficult to distinguish the characteristics of individual bacterial species and strains using only the phylum notation. Furthermore, while classification by species makes it possible to distinguish the characteristics of individual bacteria, analysis of the 16S rRNA gene by next-generation sequencers, which has a low classification rate, lacks accuracy in classification. Classification by genus allows for the distinction of individual bacterial characteristics compared to phylum, and also allows for a higher classification rate than species. For these reasons, the genus level is preferable when classifying intestinal bacterial flora.

[0056] The analysis results are obtained as the names of the classified bacteria and their numbers or occupancy rates, but simply listing them randomly does not increase their usefulness. Therefore, the results can be displayed as multiple outputs from various perspectives. One output format is to show the names of bacteria classified into 10 genera in descending order of the number of bacteria contained in feces, along with their characteristics and functionality, because it is believed that the effects of the top-ranking bacteria on the human body are most strongly reflected.

[0057] Another output format is to display a list of bacteria found in feces that are considered to be beneficial and harmful, along with their characteristics and functionality, so that the names of the beneficial and harmful bacteria that an individual possesses can be known. Yet another form of output is to classify the types and amounts of bacteria present into several categories with predetermined evaluations, and to display the categories and their meanings.

[0058] Although the types and abundance of bacteria that make up the intestinal flora vary from person to person, they can be divided into several types based on certain characteristics, and identifying the characteristics of each type is useful for the initial evaluation of the intestinal flora. One example is the classification system that divides the intestinal flora into five types: BA, BF, F, R, and P.

[0059] These five types are classified according to the species that occupy the most percentage of intestinal bacteria. Type BA is a type that is rich in Bacteroides bacteria, which are known to have beneficial effects on lipid metabolism. Type BF is a type that is rich in Bifidobacterium bacteria, which produce acetic acid and lactic acid, maintain a low intestinal pH, and suppress bacteria such as E. coli to regulate the intestinal environment. Type F is a type that is rich in Faecalibacterium bacteria, which produce butyric acid and are said to contribute to disease prevention and improved bowel movements. Type R is a type that is rich in Ruminococcus bacteria, which are said to have the function of breaking down water-soluble dietary fiber and producing short-chain fatty acids related to immunity. Type P is a type that is rich in Prevotella bacteria, which break down dietary fiber and produce succinic acid and acetic acid.

[0060] Another output format is a list of the names of bacteria present in the feces, classified by "genus" or "species," in descending order of abundance. By listing all analyzed bacteria, it is possible to display all bacteria, including those with unknown characteristics and those present in small numbers. Alternatively, based on the analytical data accumulated so far on bacterial flora, short-chain fatty acids, etc., it is also possible to display the abundance ratio of some beneficial bacteria and harmful bacteria in feces, as well as short-chain fatty acids. Such data can be used to improve an individual's intestinal environment and diet.

[0061] In this way, by analyzing the intestinal flora of an individual's feces, it is possible to obtain data on the types and amounts of bacteria contained in the intestinal flora of that individual. This data will serve as information for the use of intestinal bacteria, which will be explained next. That is, the next step is to isolate and preserve the desired beneficial bacteria from the bacteria found to be present in the intestines of the individual through bacterial flora analysis.

[0062] 3.Explanation of how to use intestinal bacteria

[0063] A method for utilizing intestinal bacteria (intestinal bacteria bank), which involves collecting and storing useful microorganisms from an individual's intestinal flora, is outlined below using the flowchart shown in Figure 1.

[0064] (1) Fecal collection This is the process of collecting the bacteria that make up the human intestinal flora (ST1 in Figure 1). To do this, human feces are collected. The collected feces are stored in a designated feces collection kit containing a designated bacterial preservative solution and then delivered to a bacterial isolation and culture facility.

[0065] (2) Bacterial flora analysis (bacterial flora analysis process) This is the process of analyzing the types and composition ratios of bacteria collected from feces (ST2 in Figure 1). By analyzing the bacterial flora of samples obtained from feces, it is possible to analyze whether or not the bacteria possessed by an individual contain certain beneficial bacteria, and in what amounts.

[0066] As explained in 1 above, this bacterial flora analysis can be carried out separately in advance, and the bacterial flora analysis carried out at this stage can be omitted.

[0067] (3) Isolation of useful bacteria This is the process of isolating the desired beneficial bacteria from the fecal fluid that has been stored in the fecal collection kit for a specified period of time (ST3 in Figure 1). Since there are various species of bacteria in the feces, including both beneficial and harmful bacteria, beneficial bacteria are sought out and identified from among them.

[0068] (4) Cultivation of isolated bacteria (cultivation process) This is the process of culturing the isolated useful bacteria (ST4 in Figure 1). The useful bacteria whose species have been identified are cultured and increased to an appropriate number.

[0069] (5) Selection of stock bacteria This is the process of selecting the most suitable strain from the same cultured species (ST5 in Figure 1). This is to collect the best strains taking into consideration factors such as ease of growth and resistance.

[0070] (6) Preservation of selected bacteria (storage process) This is the process for preserving the desired selected bacteria (ST6 in Figure 1). The selected bacteria are mixed with a preservative solution, placed in a designated tube, and frozen for storage, so that they can be taken out and used when needed.

[0071] (7) Use of preserved bacteria (production process of useful bacteria-based substances) This is the process of utilizing the preserved useful bacteria (ST7 in Figure 1). The frozen bacteria are thawed, cultured, and grown for various uses. Various substances utilizing the useful bacteria are produced, such as capsules, tablets, and other supplements containing the useful bacteria themselves, extracts from the useful bacteria, or metabolic products of the useful bacteria. These substances are then provided to the individual from whom the useful bacteria were obtained, or to others who wish to grow such useful bacteria.

[0072] (8) Providing information on intestinal flora This is the process of providing information about intestinal bacteria (ST8 in Figure 1). The characteristics of an individual's intestinal flora are identified through analysis based on bacterial flora analysis, and this information is provided and utilized. For example, by analyzing the initial analysis based on bacterial flora analysis and changes in the intestinal flora after changes in diet or taking supplements, it is possible to verify the effects of changes in diet or supplements.

[0073] The method for utilizing enterobacteria (enterobacteria bank) according to one aspect of the present disclosure is carried out as described above. However, this method for utilizing enterobacteria is merely an example for explaining one embodiment, and not all of the above steps are essential. Some steps may be omitted, other steps may be added, or steps may be interchanged if no technical inconsistency occurs.

[0074] Furthermore, the method for utilizing intestinal bacteria (intestinal bacteria bank) can be configured as something that an individual performs only once, or it can also be configured as something that can be performed multiple times over time. When an individual continuously performs the method for utilizing intestinal bacteria, one or more intestinal bacteria (including intestinal microbiota and collected feces) collected from the individual at each time can be stored, and the stored samples can be used over time. In such cases, comparison of analytical information from bacterial flora analyses performed over time can be used to improve health and prevent disease.

[0075] 4. Explanation of technologies that can constitute methods for utilizing intestinal bacteria

[0076] Examples of embodiments of various technologies that can be used to realize the above-mentioned method for utilizing intestinal bacteria (intestinal bacteria bank) will be described.

[0077] <Fecal collection process> Since each individual has a different intestinal flora, it is necessary to collect the accumulated bacteria from each individual. In order to collect the bacteria from each individual (human) without any difficulty and without surgical procedures, it is preferable to collect them from the individual's feces.

[0078] (1) Fecal collection kit It is preferable to use a fecal collection kit suitable for an intestinal bacteria bank when collecting feces. The first reason is that there is a time gap between collecting the feces and collecting the bacteria in the feces, and it is necessary to prevent the bacteria from dying during that time. The second reason is that the kit must be suitable for the collection, storage, and transportation of feces. The fecal collection kit contains a bacterial preservative solution in a specified container.

[0079] Bacterial preservation solution: First, the bacterial preservative solution enclosed in the stool collection kit must preserve the bacterial flora contained in the stool and keep the bacteria alive for a predetermined period of time before moving on to the next step. This "predetermined period" refers to the period from when the stool is placed in the stool collection kit until isolation of useful bacteria begins at the testing facility. Specifically, it is one day to one month, preferably two days to two weeks, more preferably ten to fourteen days, and even more preferably one week or about one week. This is due to the number of days required to transport the collected stool to the testing facility, and also to the fact that even after the stool collection kit containing the collected stool arrives at the testing facility, it may take several days for microorganisms to be collected and cultured. Based on these considerations, the following bacterial preservative solution was developed.

[0080] This bacterial preservation solution is a buffer solution containing a reducing agent and a cryoprotectant, and its pH is adjusted to 3.5 to 5 with a pH adjuster. Bifidobacteria and lactic acid bacteria produce organic acids and therefore have a stronger resistance to acid than other bacteria, but the pH suitable for their growth is not acidic, with the optimum pH being neutral, between 7 and 8. However, the pH is adjusted to a weak acidity in order to exhibit the effects of reducing agents such as L-cysteine ​​hydrochloride, which will be described later.

[0081] The buffer solution functions to suppress the influence of pH. Specific examples of the buffer solution include, but are not limited to, phosphate buffer, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, Tris buffer, carbonate buffer, bicarbonate buffer, acetate buffer, citrate buffer, phosphate-buffered saline (PBS), HEPES-buffered saline, and Tris-buffered saline. More specifically, the buffer solution is preferably phosphate-buffered saline (PBS) or D-PBS containing potassium chloride, and more preferably Dulbecco's phosphate-buffered saline (D-PBS(-)) (also referred to as "Dulbecco's phosphate-buffered saline"), which does not contain divalent cations. Dulbecco's phosphate-buffered saline is more preferable because it minimizes pH fluctuations in the bacterial preservative solution due to the addition of feces and can maintain the viability of the bacterial cells in the feces without damaging them. The term "Dulbecco's phosphate-buffered saline, etc." in the claims may include the various buffer solutions exemplified above.

[0082] Reducing agents are components that do not inhibit the growth of anaerobic bacteria such as bifidobacteria and lactic acid bacteria, and also contribute to improving the growth of lactic acid bacteria. Examples of reducing agents include L-cysteine, N-acetylcysteine, and methylene blue. L-cysteine ​​hydrochloride is used as a food additive and also to reduce oxygen in water to create anaerobic conditions.

[0083] The pH adjuster is used to reduce stress on fecal bacteria by adjusting the strongly acidic (pH 2 or more but less than 3) bacterial preservative solution to a weakly acidic (pH 3 or more but less than 6, preferably pH 3.5 to 5) pH. Examples of pH adjusters include, but are not limited to, carbonates, bicarbonates, hydrochloric acid, and sodium hydroxide. More specifically, sodium bicarbonate and sodium carbonate are preferred pH adjusters, with sodium bicarbonate being more preferred. This is because sodium bicarbonate is suitable for the growth of anaerobic methanogens and allows for easy fine adjustment of the pH. Furthermore, while hydrochloric acid and sodium hydroxide are considered toxic substances, sodium bicarbonate is commonly used as baking soda and is therefore highly safe. The term "sodium bicarbonate, etc." in the claims may include the various pH adjusters listed above.

[0084] The cryoprotectant can impart a suitable viscosity to the preservation solution, and specific examples include, but are not limited to, glycerol and dimethyl sulfoxide (DMSO). Glycerol is also preferred because it functions as an emulsifier, stabilizer, and anti-caking agent. Another reason why glycerol is preferred is that it is cheaper than the organic solvent dimethyl sulfoxide and is not dangerous to the human body, making it widely used in bacterial preservation. Note that the term "glycerol, etc." in the claims can include the various preservation solutions exemplified above.

[0085] The bacterial preservative solution is preferably prepared so that its pH is between 3.5 and 5. This is because useful bacteria can survive if the pH of the stool collection solution after the required amount of stool is placed in a stool collection kit and mixed with the bacterial preservative solution is 5 or higher, and therefore the pH of the bacterial preservative solution may be 4 to 5, or 3.5 to 5, in order to achieve a pH of 5 or higher after mixing with stool.

[0086] In other words, if the pH is not adjusted by adding a pH adjuster, the bacterial preservation solution will be strongly acidic (pH 2 or higher but lower than 3), which is a considerable stressor for the intestinal bacteria. However, to reduce the stress on the intestinal bacteria while maintaining the effectiveness of the reducing agent, the solution is adjusted to a weak acidity (pH 3 or higher but lower than 6, preferably pH 3.5 to 5).

[0087] The content of each component in the bacterial preservation solution is 0.005 to 0.02 g of reducing agent per 10 mL of buffer and cryoprotectant combined. Less than 0.005 g will not have a reducing effect, while more than 0.02 g will result in a too low pH.

[0088] The content of the pH adjuster can be 0.5 mg to 30 mg, 0.5 mg to 10 mg, or 0.5 mg to 2 mg per 10 mL of the total buffer solution and cryoprotectant. If the content is less than 0.5 mg, pH adjustment is difficult, and if it is less than 5 mg, pH adjustment may not be possible. If the content exceeds 2 mg, 10 mg, or 30 mg, the effects of other ingredients may be reduced.

[0089] The ratio of the buffer solution and the cryoprotectant in a total of 10 mL can be adjusted appropriately within a suitable viscosity range.

[0090] A preferred embodiment of the bacterial preservative solution is a mixture of 5 mL of 50% glycerin solution, 5 mL of D-PBS(-), 0.01 g of L-cysteine ​​hydrochloride, and 0.5 mg to 10 mg, or 0.5 mg to 2 mg, of sodium bicarbonate.

[0091] Even if feces are stored in a general fecal preservative solution, the useful anaerobic bacteria often die off. However, the bacterial preservative solution described above is suitable for preserving anaerobic bacteria, and reduces the possibility of the useful bacteria dying off between the time the feces of an individual user is collected and the time it is received by a service provider, such as the operator of this intestinal bacteria bank.

[0092] In another embodiment of the bacterial preservative solution, the bacterial preservative solution further comprises a gelling agent. This bacterial preservative solution has the property of becoming a non-flowing gel when left for a predetermined period of time, and becoming a flowing liquid when mixed with feces and shaken. "A gel that does not flow when left for a predetermined period of time" refers to a gel that loses its fluidity when left at room temperature for at least one day, and changes to a fluid sol when shaken. Furthermore, when mixed with feces and shaken, the bacterial preservative solution and feces mix to form a fecal collection solution, which is a fluid sol. The fecal collection solution is in the form of mixed feces, and after shaking, it may become a gel or remain in a sol state.

[0093] The amount of gelling agent added (gelling agent amount) is preferably 0.05 to 0.35% (w / v) of the bacterial preservative solution, and more preferably 0.075 to 0.25% (w / v). The reason for this range is that if the gelling agent is added at a concentration below 0.05% (w / v), the liquid state will not produce its gelling effect, and if the gelling agent is added at a concentration above 0.35% (w / v), it will be difficult to uniformly mix the gelling agent with the feces. On the other hand, if the gelling agent is added at a concentration within the range of 0.05 to 0.35% (w / v), the feces and the bacterial preservative solution can be easily mixed by simply shaking the container containing the feces and the bacterial preservative solution about 10 times, and the preservation of the bacteria contained in the feces is improved. Furthermore, the handling and operability of the feces collection kit equipped with the bacterial preservative solution are improved. Furthermore, a concentration within the range of 0.075 to 0.25% (w / v) also promotes the growth of Bifidobacteria.

[0094] Gel-type bacterial preservative solutions are superior to liquid-type bacterial preservative solutions in preserving difficult-to-culture bacteria. In other words, feces contain fewer bacteria than easily cultured bacteria such as Bifidobacterium, making it possible to culture certain bacteria that are difficult to isolate and culture later. While the reason for this is unclear, it is speculated that the gel-type rather than liquid-type bacterial preservative solution allows bacteria to undergo gentler changes when exposed to the changing environment from feces to the bacterial preservative solution. Furthermore, the inclusion of a gelling agent is thought to disperse and stabilize the buffer, reducing agent, cryoprotectant, and pH adjuster contained in the bacterial preservative solution, thereby improving bacterial preservation.

[0095] Examples of gelling agents include gellan gum, carrageenan, carboxymethylcellulose, sodium alginate, sodium polyacrylate, xanthan gum, locust bean gum, pectin, and gelatin, with gellan gum being preferred. Gellan gum is a gelling agent derived from Pseudomonas elodea ( Pseudomonas elodea Gellan gum is a polysaccharide produced by the bacterium Glycosphaerella glabrata (Gellan gum), which has been deacetylated and purified. Its main components are glucose, rhamnose, and uronic acid, and it may be suitable for culturing microorganisms that are difficult to culture. In contrast, when agar is used as a gelling agent, it can inhibit the swarming of certain microorganisms, hindering their cultivation. The reason why gellan gum is suitable for culturing microorganisms that are difficult to culture is not only because the bacterial preservative solution has a moderate viscosity, which acts gently on the bacterial environment, but also because it acts on extracellular enzymes involved in the uptake of nutrients, thereby providing a supporting effect on difficult-to-cultivate bacteria.

[0096] container: The stool collection kit consists of a container and a bacterial preservative solution contained within it.

[0097] The container preferably comprises a container body and a lid. The container body is made of glass or plastic, has a bottom, an opening at the top, and is capable of enclosing the bacterial preservative solution. The lid seals the opening of the container body, and preferably has a spoon-shaped spatula attached to the inside of the lid for scraping stool and depositing it into the container body. The spatula preferably has a recess that can hold approximately 1 to 2 g of stool. This is because the desired amount of stool, 1 to 2 g, can be collected by scraping stool with the tip and depositing it on the tip. However, since a fairly large recess is required to hold 1 to 2 g of stool, the size of the recess may be set to a size that can hold about half or one-third of 1 to 2 g of stool, allowing for two or three stool collections.

[0098] The container consisting of the container body and the lid may further include an outer container for enclosing the container. By providing the outer container, leakage of the stool collection liquid to the outside can be more effectively prevented.

[0099] An example of a stool collection kit is shown in the schematic perspective view of Figure 3. This stool collection kit 10 has an inner container 20 and an outer container 30 that encloses the inner container 20. The inner container 20 is composed of an inner container body 21 that contains a bacterial preservative solution 40, and a lid 22 that seals the bacterial preservative solution 40 inside the inner container body 21. A spoon-shaped spatula 23 attached to the lid 22 has a recess 25 at the tip of its handle 24 for placing feces thereon, and a baffle plate 26 near the recess 25 on the handle 24 to allow the feces to be suitably mixed into the bacterial preservative solution 40. The recess 25 may be shaped to be bent from the handle 24, or it may be shaped to extend straight from the handle 24.

[0100] The outer container 30 is a container that encloses the inner container 20, and is composed of an outer container body 31 and a lid 32. A shielding film 33 is provided on the inner surface of the outer container body 31 so that the contents cannot be seen from the outside. Because the inner container 20 is placed inside the outer container 30, even if the fecal collection liquid, in which feces is mixed with the bacterial preservative solution 40, leaks outside the inner container 20, leakage from the outer container 30 can be prevented, making it hygienic and preventing the smell of feces from leaking. The inner container 20 and the outer container 30 may be commercially available containers manufactured by Sarstedt or the like.

[0101] It is preferable to place a predetermined number of stirring balls 27 in the inner container 20. Since the bacterial preservative solution 40 becomes gelatinous and loses fluidity after being left for a while, it is preferable to shake the inner container 20 to mix the feces after adding it, and the presence of stirring balls 27 in the inner container 20 helps to fluidize the bacterial preservative solution, making it easier to mix the bacterial preservative solution with the feces. The stirring balls 27 may be any suitable ball that contributes to mixing the bacterial preservative solution with the feces and promotes mixing of the feces and bacterial preservative solution. However, stainless steel balls with a diameter of 2 to 4 mm are preferred, and it is preferable to place 3 to 10 of them in the inner container 20. The stainless steel balls do not change the properties of the collected feces liquid, and placing 3 to 10 balls with a diameter of 2 to 4 mm can improve stirring efficiency. Balls smaller than 2 mm, larger than 4 mm, or fewer than 3 balls will result in poor stirring efficiency. Furthermore, placing balls larger than 4 mm or more than 10 balls will result in a relatively small volume of the bacterial preservative solution 40, which is undesirable as it increases costs. One embodiment of using stirring balls 27 is to use 4, 5, 6, 7, 8, or 9 balls with a diameter of 3 mm.

[0102] The amount of feces collected is 0.5 to 3 g, preferably 1 to 2 g. If the amount is less than 0.5 g, the necessary beneficial bacteria may not be collected. Furthermore, since the necessary beneficial bacteria can be collected with an amount up to 3 g, collecting feces in excess of 3 g increases the amount of bacterial preservative solution required and requires a larger inner container, resulting in higher costs. The volume of bacterial preservative solution included in the stool collection kit is 5 to 20 mL, preferably 5 to 15 mL, and more preferably 8 to 12 mL. This is because it is necessary to include the amount necessary to mix with the stool to be collected and obtain the bacteria therein without killing them. If it is less than 5 mL, it may be too small for the amount of stool to be collected, and beneficial bacteria in the stool may die. If it exceeds 20 mL, the inner container will become larger and more expensive.

[0103] The inner container preferably contains 30 to 90 vol% of the bacterial preservative solution. If the volume is less than 30 vol%, the protection of bacteria in the feces may be insufficient, and the amount of voids that may be mixed into the inner container when the feces are mixed in may increase, potentially impairing the survival of anaerobic bacteria. If the volume is more than 90 vol%, the fecal collection solution may overflow from the inner container when the feces are placed in the inner container. Therefore, it is preferable to select and use an inner container with an internal volume of 5.5 to 23 mL, preferably 12 to 18 mL, so that the above-mentioned amount of collected feces can be placed in the inner container containing the bacterial preservative solution with the above proportion and the inner container can be sealed.

[0104] After collecting the stool, it is preferable to shake the inner container 20 containing the stool 5 to 20 times, preferably 8 to 12 times, to thoroughly mix the stool with the bacterial preservative solution. This is because the beneficial bacteria may die if the stool is not sufficiently enveloped in the bacterial preservative solution and comes into contact with air. The outer container 30 may not be required.

[0105] The stool collection kit containing the stool is then packaged and delivered from the individual to a designated facility where bacteria will be isolated. It is preferable to store the stool collection kit in a refrigerator between collection and delivery. Since it is desirable to extract bacteria from the stool as soon as possible, it is preferable to send the kit refrigerated for no more than two days after collection.

[0106] <Isolation process of beneficial bacteria> This is a process for isolating specific beneficial bacteria from the many types of bacteria contained in feces.

[0107] Beneficial bacteria are bacteria that have beneficial effects on human health, such as the so-called good bacteria. Examples of beneficial bacteria include bifidobacteria and lactic acid bacteria. Among lactic acid bacteria, Lactobacillus ( Lactobacillus ) may be present in small numbers in feces depending on the individual. Therefore, isolation of Lactobacillus is effective for individuals whose bacterial flora has been analyzed using next-generation sequencing (NGS) and whose presence has been confirmed.

[0108] Other useful bacteria include the genus Faecalis bacterium ( Faecalibacterium ) and Roseburia spp. ( Roseburia ) and Akkermansia spp. ( Akkermansia ), Collinsella spp. ( Collinsella ), Pediococcus spp., Blautia spp. Blautia ) and other bacteria other than lactic acid bacteria.

[0109] To isolate useful bacteria, it is preferable to use a liquid medium for the fecal fluid that corresponds to the bacteria to be isolated. This is because the isolation of the desired bacteria becomes easier if the growth of bacteria other than the desired bacteria can be eliminated. Such media include media that allow the growth of bacteria other than the target bacteria to be grown and to which a selective agent such as an antibiotic that inhibits the growth of other bacteria is added, media that use a carbon source that only the desired bacteria can utilize to obtain colonies of only the desired bacteria, and media that utilize properties unique to the desired bacteria.

[0110] When Bifidobacterium is isolated using a liquid medium selected according to the bacterium, examples of the liquid medium include MGLP agar medium, modified BCP-supplemented plate count agar medium, TOS mupirocin medium, TOS propionic acid agar medium, and paromomycin-containing TOS propionic acid agar medium. MGLP agar medium and modified BCP-added plate count agar medium can suppress the growth of lactic acid bacteria, which are prone to contaminating bacteria, by limiting the nutrient source. In TOS mupirocin medium, bifidobacteria grow well due to the galactooligosaccharides in the TOS medium, while mupirocin inhibits the growth of lactic acid bacteria, preventing the inclusion of lactic acid bacteria, which tend to coexist with bifidobacteria.

[0111] TOS propionic acid agar medium contains galactooligosaccharides that are excellent at selectively promoting the growth of bifidobacteria, and while propionic acid has a growth inhibitory effect on lactic acid bacteria, it has a growth promoting effect on bifidobacteria. By further adding paromomycin to the TOS medium, the paromomycin-containing TOS propionic acid agar medium can inhibit protein synthesis of highly sensitive harmful bacteria, thereby preventing contamination by harmful bacteria.

[0112] When isolating lactobacilli, MRS medium or MRS medium containing bile acid (cholic acid) can be used. MRS medium contains the nutrients required by fastidious lactic acid bacteria, and the ammonium citrate and sodium acetate it contains inhibit the growth of many bacteria other than lactic acid bacteria. By using MRS medium containing bile acids, lactic acid bacteria with high resistance to bile acids can be obtained, and lactic acid bacteria that can easily reach the intestines can be selectively obtained.

[0113] Alternatively, instead of using a selective medium that does not allow the growth of bacteria other than those specified as useful bacteria, a medium in which various bacteria can grow can be used for cultivation, and then, from all the resulting colonies, DNA can be extracted from a colony that has the characteristics of the desired useful bacteria, and the bacterial species can be identified to isolate the desired useful bacteria. By using a medium in which multiple bacterial species can grow, the effort of culturing in multiple media can be eliminated.

[0114] Examples of such media include M2GSC medium, BCP-supplemented plate count agar medium (BCP plate medium), and calcium carbonate-containing MRS agar medium. M2GSC medium is used to cultivate bacteria that are difficult to culture or highly oxygen-sensitive, such as Faecalibacterium, but it also allows Lactobacillus to grow because it contains high nutritional sources for Lactobacillus, such as glucose, fructose, and cellobiose. BCP-added plate count agar medium makes it easy to detect lactic acid bacteria even when it contains a variety of bacteria, because the BCP (bromcresol purple) contained in the medium turns yellow when lactic acid is produced by lactic acid bacteria. Calcium carbonate-containing MRS agar medium is cloudy due to the calcium carbonate it contains, but lactic acid bacteria dissolve the calcium carbonate, making the area around the lactic acid bacteria colonies transparent and making it easier to detect the lactic acid bacteria.

[0115] After culturing in these media, the desired useful bacteria can be isolated by extracting DNA from the resulting colonies and identifying the bacterial species.

[0116] <Selection process for isolated bacteria> Since bacteria belonging to the same genus have different characteristics depending on the strain, it is important to select useful bacteria to be stored even if they belong to the same Bifidobacterium or Lactobacillus in order to obtain useful bacteria with a high degree of usefulness. For example, if a strain with high bile acid resistance is identified by performing a bile acid resistance test on multiple isolated strains, it is believed that the bacterium will also have high resistance to gastric acid and be more likely to reach the intestine. In order to obtain bacteria with stronger desirable properties, various selection steps such as the bile acid resistance test can be added.

[0117] <Preservation (storage) process of selected bacteria (process of enriching isolated bacteria)> The isolated and identified cells can be mixed with a culture medium and a preservative solution, placed in a designated tube, and frozen for storage, thereby preserving useful bacteria. The tube to be stored is preferably a plastic container suitable for frozen storage. For convenience of storage, a tube having a capacity of about 1.0 to 5.0 mL can be used, and a capacity of 1.5 to 2.0 mL is preferred.

[0118] Various media, such as MRS medium, can be used as the culture medium containing the useful bacteria before mixing with the cryoprotectant. It is preferable that the medium contains the isolated useful bacteria at a concentration of 1.00E+08 / mL or more, or at a concentration up to the point where the growth curve reaches stationary phase. This is because stationary phase cells are thought to be more resistant to freezing stress than logarithmic growth phase cells. Furthermore, because a certain percentage of cells die upon freezing, a concentration below 1.00E+08 / mL may pose a risk of difficulty in growing the bacteria to the required amount upon use, resulting in poor preservation of the useful bacteria. Furthermore, it is practically difficult to contain the useful bacteria at a concentration significantly exceeding 1.00E+08 / mL.

[0119] The cryoprotectant content in the mixture obtained by mixing this culture medium containing beneficial bacteria with a cryoprotectant is 15 to 20 vol%. If the cryoprotectant content is less than 15 vol%, the culture medium may freeze during freezing, potentially destroying the beneficial bacteria. On the other hand, if the cryoprotectant content is more than 20 vol%, the percentage of beneficial bacteria in the liquid will decrease, making it difficult to grow these beneficial bacteria when used, and preservation efficiency will be poor.

[0120] After mixing the medium containing beneficial bacteria with a cryoprotectant, the resulting storage solution can be stored in tubes under several conditions: using liquid nitrogen at −196°C to −160°C, or in an ultra-low temperature freezer rated for −150°C, −90°C to −80°C, −60°C to −40°C, or −35°C to −20°C. Lower temperatures have the advantage of halting bacterial metabolic activity and maintaining the cells in a dormant state for longer periods. However, temperatures between −196°C and −150°C are costly and require extensive space for maintaining the temperature. Furthermore, temperatures between −60°C and −20°C are susceptible to the effects of ambient temperatures, and large fluctuations in the temperature inside the storage cabinet when it is opened or closed can lead to deterioration in the quality of frozen samples, making long-term storage difficult. Therefore, it is preferable to store samples frozen at around −80°C in an ultra-low temperature freezer rated for −90°C to −80°C.

[0121] <Use of the intestinal bacteria bank> The preserved beneficial bacteria are cultured, and the beneficial bacteria themselves, extracts from the beneficial bacteria, or metabolic products of the beneficial bacteria are encapsulated or used as ingredients in supplements to produce beneficial bacteria-related products. For example, by returning the bacteria to the individual from whom they were collected, they can contribute to improving the intestinal flora of that individual.

[0122] Alternatively, providing the above-mentioned capsules, supplements, etc. derived from the banked specific beneficial bacteria to individuals who do not have the specific beneficial bacteria or who have them in small numbers can help improve the intestinal flora of those individuals. [Example]

[0123] <Experiment 1: Functional evaluation of bacterial preservative solution (part 1)> Test purpose: To evaluate the preservation of bifidobacteria in a fecal collection solution so that beneficial bacteria in the collected feces do not die out after a predetermined time has passed in the fecal collection solution.

[0124] Preparation of bacterial stock solution: Six bacterial preservative solutions were prepared by mixing D-PBS(-), glycerin solution, L-cysteine ​​hydrochloride, and sodium bicarbonate solution. The concentrations were 50% (v / v) of D-PBS(-), 0.1% (w / v) of L-cysteine ​​hydrochloride, and 25% (v / v) of glycerin, with pH values ​​of 3.0, 3.5, 4.0, 4.5, 5.0, and 5.5. These solutions were designated Samples 2 to 7. A bacterial preservative solution without added sodium bicarbonate (NC) was also prepared and designated Sample 1. The pH of this unadjusted bacterial preservative solution was strongly acidic, ranging from 2.7 to 2.8.

[0125] Test Method: Approximately the same amount of feces from the same person defecated at the same time was added to 10 mL of the above sample, and the mixture was completely suspended until all fecal masses disappeared. -3 , 10 -4 , 10 -5 The final dilution was applied to TOS medium, a specialized plate medium for Bifidobacterium, and cultured for 72 hours at 37°C under anaerobic conditions. After culture, the number of colonies formed on the plate was counted, and the survival rate was calculated by applying the following formula to the number of colonies on the day of collection and after 7 days of storage. Viability = (number of colonies after 7 days of storage / number of colonies on the day of collection) In this test, samples were prepared in the same manner on different stool collection days (defecation days), and the number of samples for each was four.

[0126] Test results: The viability of each of the above samples is shown in the box plot in Figure 2. As shown in Figure 2, for samples 3 to 6, which had a pH range of 3.5 to 5.0, the median was close to the standard value of 1.0 for viability, and the interval between the maximum and minimum values ​​was narrow. Furthermore, compared to sample 1, whose pH was not adjusted, and sample 7, whose pH was 5.5, the four viability results for these samples were concentrated in the middle.

[0127] On the other hand, sample 1, which had not been adjusted for pH, sometimes had a survival rate of 0, and the median was the furthest from the standard value of 1.0. Sample 7, which had a pH of 5.5, had the highest survival rate compared to the other samples, but this was thought to be due to the proliferation of bacteria other than Bifidobacterium.

[0128] These results confirmed that by using a bacterial preservative solution with a pH adjusted to 3.5 to 5, bifidobacteria can be left in the fecal sample for a specified period of time without being killed.

[0129] <Experiment 2: Isolation and culture of Lactobacillus> Test Purpose To investigate suitable methods for isolating lactobacilli.

[0130] Test Method We attempted to isolate Lactobacillus using M2GSC medium and MRS medium containing bile acid (cholic acid). The same fecal sample solution (sample 5a) mixed with the same feces as used in Experiment 1 (pH 4.5 after preparation of bacterial preservative solution, pH 6.39 after mixing with feces) was added at 5.0% (v / v) to M2GSC medium, MRS medium, and MRS medium containing 0.05-1.0% (w / v) bile acid, and cultured at 37°C under anaerobic conditions for 72 hours.

[0131] After culturing, the culture medium was -3 , 10 -4 , 10 -5 The culture was serially diluted 1:1, and the final dilution was applied to MRS medium. It was then cultured at 37°C under anaerobic conditions for 48 hours. Since Lactobacillus colonies are primarily round, yellow, or round, white, six colonies formed on the plate were selected. DNA was extracted from the six colonies, and the 16S rRNA gene (1.5 kb) was amplified by PCR. The nucleotide sequence of the amplified PCR product was analyzed using a sequencer, and the bacterial species were identified using the NCBI Blast search system.

[0132] Test results: Round yellow or round white colonies were observed on all three media. However, the number of round yellow colonies was lower on regular MRS medium than on M2GSC medium and MRS medium containing bile acids.

[0133] The bacteria obtained from the six colonies selected from M2GSC medium and standard MRS medium all showed high homology (98% or more) with Lactobacillus species. However, the species obtained were biased depending on the medium. In M2GSC medium, only Lacticase Bacillus paracasei ( Lacticaseibacillus paracasei ) or Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ) preferentially grows, and in the normal MRS medium, Rimosilactobacillus fermentum ( Limosilactobacillus fermentum ) was found to grow preferentially.

[0134] Needle-shaped crystals were observed after cultivation in the bile acid-containing MRS medium. These crystals are metabolic products produced by the decomposition of bile acids by bile salt hydrolase (BSH) possessed by lactic acid bacteria such as Lactobacillus. BSH-producing lactic acid bacteria are known to promote the reduction of blood cholesterol, and it was found that such lactic acid bacteria grow in the bile acid-containing MRS medium. In addition, Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ), Lactipranchi Bacillus pentosus ( Lactiplantibacillus pentosus ), Lactobacillus seniori ( Lentilactobacillus senioris ), Lactipranchi Bacillus fabifermentans ( Lactiplantibacillus fabifermentans Lactic acid bacteria such as Lactobacillus plantarum were also found, with Lactobacillus plantarum being particularly abundant.

[0135] <Experiment 3: Isolation and culture of Bifidobacterium> Test Purpose To investigate suitable methods for isolating Bifidobacteria.

[0136] Test Method A paromomycin-containing TOS propionic acid agar medium suitable for the growth of Bifidobacterium was prepared in advance as follows.

[0137] First, paromomycin sulfate was dissolved in distilled water to a concentration of 10 mg / mL, and the solution was sterilized by filtration using a 0.22 μm syringe filter. Next, TOS agar medium was autoclaved (115°C, 15 minutes) and then cooled to 50°C. The paromomycin sulfate solution was added to the TOS agar medium to a final concentration of 50 μg / mL and mixed.

[0138] Next, the same fecal collection fluid as used in Experiment 2 was diluted with D-PBS(-) for 10 min. -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 The strain was serially diluted 2-fold, and 80 μL of the final dilution was spread onto the paromomycin-containing TOS propionic acid agar medium. The medium was then cultured at 37°C under anaerobic conditions for 48 to 72 hours. DNA was extracted from the single colonies that formed, and the 16S rRNA gene (1.5 kb) was amplified by PCR. The nucleotide sequence of the amplified PCR product was analyzed by sequencer, and the bacterial species was identified using NCBI's Blast search system.

[0139] Test results: The bacteria identified from the single colonies showed high homology (98% or more) with species of the genus Bifidobacterium.

[0140] <Experiment 4: Preservation (storage) of selected bacteria> Test Purpose To prepare a storage tube for storing the desired beneficial bacteria obtained by collecting, isolating, culturing, and selecting them from feces.

[0141] The useful bacteria were stored in 1.5-2.0 mL tubes for frozen storage. First, colonies of beneficial bacteria selected from those isolated from feces and cultured in pure culture were scraped with a platinum loop and inoculated into MRS medium, where they were further cultured at 37°C for 48 to 72 hours under anaerobic or aerobic conditions. The anaerobic or aerobic conditions were determined according to the type of beneficial bacteria. To the tube, 200 μL of a sterilized 50% glycerol solution was added, and then 800 μL of the culture medium of the beneficial bacteria was added and mixed. An IC chip was attached to the tube, and the obtained useful bacteria were stored by freezing at a temperature of ≦−80°C.

[0142] <Experiment 5: Functional evaluation of bacterial preservative solution (part 2)> Test Purpose As explained in the section on the prior art, a preservation solution for preserving and transporting various bacteria contained in human feces (hereinafter referred to as the "conventional preservation solution") is described in Patent Document 3 (US Patent Application Publication No. 2022 / 0160337). However, because this conventional preservation solution has a pH of 7.5, its ability to preserve anaerobic bacteria in feces is questionable. Therefore, the preservation performance of Akkermansia, a useful enterobacterium and an obligate anaerobe, was examined for the bacterial preservation solution of the present invention and the conventional preservation solution.

[0143] (1) Akkermansia ( Akkermansia muciniphila (JCM strain) ) was cultured under anaerobic conditions at 37°C for 72 to 96 hours.

[0144] Modified GAM liquid medium (composition): Modified GAM bouillon...41.70g / L Sodium propionate...0.60g / L N-acetyl-D(+)-glucosamine: 0.11g / L Agar (used when preparing plate medium)...15.00g / L

[0145] (2) The bacterial preservative solution of the present invention had the following composition and was designated Sample 8. Bacterial preservative solution of the present invention (composition): L-cysteine ​​hydrochloride...1g / L 50% (w / v) glycerol...500mL D-PBS...500mL NaHCO3...as needed (add until pH reaches 4.0)

[0146] On the other hand, the conventional preservation solution used was the preservation solution described in Table 1 of the specification of Patent Document 3. The composition is as follows, and this was designated Sample 9. Conventional preservative solution (composition): Sodium thioglycolate...1g / L Na2HPO4...1.15g / L NaCl……3g / L KCl……0.2g / L KH2PO4……0.2g / L MgSO4·7H2O……0.1g / L L-cysteine: 1g / L Glycerol...200mL Activated carbon...1g / L Antioxidant enzyme (superoxide dismutase, derived from bovine erythrocytes (Cu / Zn type))...600U Sterile water...800mL The pH of the conventional preservative solution having the above composition was 7.5.

[0147] (3) After culturing the Akkermansia, the supernatant and Akkermansia cell pellet were separated by centrifugation, and the supernatant was removed. After removing the supernatant, the cell pellet was washed once with D-PBS, centrifuged, and the supernatant was removed. The cell pellet was dissolved in 10 mL of D-PBS to prepare an Akkermansia suspension. This Akkermansia suspension was added to 10 -3 , 10 -4 , 10 -5 After serial dilutions, 80 μL of each dilution was added and spread onto modified GAM plates and cultured at 37°C for 72 to 96 hours under anaerobic conditions. After culture, the colonies formed on the plates were counted, and the viable number of Akkermansia bacteria in the suspension was calculated.

[0148] (4) 500 μL of the Akkermansia suspension prepared in (3) above was added to 5 mL of each of Sample 8, the bacterial preservative solution of the present invention prepared in (2) above, and Sample 9, the conventional preservative solution, and mixed, and stored at 4°C for 5, 7, and 10 days. After storage, these mixtures were -3 , 10 -4 80 μL of each diluted solution was added and spread onto modified GAM plates and cultured at 37°C under anaerobic conditions for 72 to 96 hours. After culture, the colonies formed on the plates were counted, and the number of viable Akkermansia bacteria in each mixture was calculated.

[0149] The ratio of the viable cell count cfu in (4) above to the viable cell count cfu in (3) above was taken as the survival rate of Akkermansia for each day that had passed. That is, the viability (%) was calculated as follows: number of viable bacteria (cfu) after storage at 4°C (5, 7, and 10 days) × 9.09 / number of viable bacteria (cfu) in 500 μL of Akkermansia suspension × 100. The reason for multiplying by 9.09 is that 500 μL of Akkermansia suspension was added to 5 mL of storage solution, so the Akkermansia suspension was diluted 9.09 times (0.5 mL / 5.5 mL × 100 = 9.09 times).

[0150] result The viable cell counts (cfu) of Sample 8, the bacterial preservative solution of the present invention, and Sample 9, the conventional preservative solution, were 2.12E+07 on day 0, 1.22E+07 for Sample 8 and 3.49E+06 for Sample 9 on day 5, and 4.36E+06 for Sample 8 and 1.90E+06 for Sample 9 on day 10. From this, the survival rates were as follows: Sample 8: 100% (day 0), 57.4% (day 5), 20.6% (day 10) Sample 9: 100% (day 0), 16.5% (day 5), 8.9% (day 10)

[0151] Consideration From the results of the survival rate of Akkermansia, Sample 8, the bacterial preservative solution of the present invention, had a higher number of live Akkermansia bacteria and survival rate than Sample 9, the conventional preservative solution. The reason for this is thought to be that the bacterial preservative solution of the present invention is adjusted to an acidic pH of 4 to enhance the oxygen-scavenging effect of the reducing agent, L-cysteine ​​hydrochloride, whereas the pH of conventional preservative solutions is adjusted to a weak alkaline pH of 7.5, making it impossible to maintain the reducing effect of L-cysteine ​​hydrochloride in the preservative solution.In addition, the bacterial preservative solution of the present invention uses NaHCO3 when adjusting the pH, which is thought to produce a synergistic effect of oxygen-scavenging action with L-cysteine ​​hydrochloride.

[0152] Incidentally, Akkermansia is listed in Fig. 7D of Patent Document 3 (US Patent Application Publication No. 2022 / 0160337), but this represents the abundance ratio of Akkermansia OTUs contained in Total OTUs (operational taxonomic unit: essential bacterial genes (generally 16S ribosomal RNA genes)) in the feces, and indicates the gene amount of both live and dead bacteria. Therefore, it is unclear how much live Akkermansia is contained, and this description in Patent Document 3 does not explain the survival of useful bacteria.

[0153] <Experiment 6: Preparation of gel-like bacterial preservative solution and viscoelasticity measurement> Preparation of bacterial stock solution: D-PBS(-), glycerin solution, L-cysteine ​​hydrochloride, gellan gum, and sodium bicarbonate solution were mixed. Specifically, gellan gum powder was added to the glycerin solution and heated to dissolve the gellan gum. Meanwhile, L-cysteine ​​hydrochloride was dissolved in D-PBS(-) and sodium bicarbonate solution was added, and this solution was mixed with the glycerin solution containing gellan gum at a 1:1 ratio (volume). Six bacterial preservation solutions were prepared: 50% (v / v) D-PBS(-), 0.1% (w / v) L-cysteine ​​hydrochloride, 25% (v / v) glycerin, and varying gellan gum concentrations, all at pH 4. The gellan gum concentrations and sample numbers for these six bacterial preservation solutions are as follows: The gellan gum concentrations were sample 11 at 0.02% (w / v), sample 12 at 0.075% (w / v), sample 13 at 0.1% (w / v), sample 14 at 0.3% (w / v), sample 15 at 0.35% (w / v), and sample 16 at 0.40% (w / v).

[0154] Visual evaluation Sample 11, which had the lowest gellan gum concentration of 0.02% (w / v), was more viscous than the sample without gellan gum, but remained liquid and did not gel. On the other hand, Samples 12 to 16 formed gels that did not flow or flow even when the tubes containing the samples were tilted. Furthermore, when feces were added to the bacterial preservative solutions of Samples 12 to 15, the feces mixed with the bacterial preservative solution, whereas when feces were added to the bacterial preservative solution of Sample 16, the feces did not mix completely with the bacterial preservative solution.

[0155] Viscoelasticity measurement (1)...Strain dispersion For the above samples 12 and 15, a strain dispersion test (storage modulus (G') and loss modulus (G") were measured at 30°C and 1.0 Hz within a strain range of 0 to 1000%) was carried out using a rheometer MCR101 (manufactured by Anton Paar). As a result, for sample 12, which had a gellan gum concentration of 0.075% (w / v), the storage modulus (G') was in the range of 2.42E+00 to 2.60E+00 and the loss modulus (G") was in the range of 3.60E-01 to 4.26E-01 at strains between 3.98% and 39.8%. From this, it was determined that the change in both moduli was small between 3.98% and 39.8%, and they were in the linear region, which is a stable region. On the other hand, for sample 15, which had a gellan gum concentration of 0.35% (w / v), the storage modulus (G') was in the range of 1.00E+03 to 1.03E+03 and the loss modulus (G") was in the range of 8.24E+01 to 9.11E+01 at strains between 0.0626% and 1.58%. From this, it was determined that the change in both moduli was small between 0.0626% and 1.58%, and they were in the linear region, which is a stable region.

[0156] Viscoelasticity Measurement (2)...Frequency Dispersion For the above-mentioned Samples 12 and 15, strain (%) was selected from the linear region based on the results of the strain dispersion test, and the frequency dispersion was measured. That is, for Sample 12, the changes in storage modulus and loss modulus were measured at 30° C., with a strain (%) of 10%, and by changing the frequency between 0.6 and 62.8 Hz. As a result, both the storage modulus and the loss modulus increased as the frequency increased, and the storage modulus increased sharply when the frequency exceeded 10.0 Hz. Since the storage modulus was greater than the loss modulus at any frequency within the above range, Sample 12 is considered to be a gel-like substance based on the results of the frequency dispersion test. Furthermore, for Sample 15, the changes in storage modulus and loss modulus were measured at 30° C., with a strain (%) of 0.16%, and by changing the frequency between 0.6 and 62.8 Hz. As a result, the storage modulus increased as the frequency increased, while the loss modulus remained almost constant. Since the storage modulus was greater than the loss modulus at any frequency within the above range, sample 15 is considered to be a gel-like material, based on the results of the frequency dispersion test.

[0157] Viscoelasticity Measurement (3)...Temperature Dispersion For the above-mentioned Samples 12 and 15, strain (%) was selected from the linear region based on the results of the strain dispersion test, and the temperature dispersion was measured. That is, for Sample 12, the changes in storage modulus and loss modulus were measured by changing the temperature between 2.55 and 60°C at a frequency of 1.0 Hz and a strain (%) of 10%. As a result, the storage modulus decreased with increasing temperature, and the material tended to become a liquid (sol). Furthermore, since the storage modulus was greater than the loss modulus at any temperature within the above range, sample 12 is considered to be a gel-like substance from 2.55 to 60°C, based on the results of the temperature dispersion test.

[0158] For Sample 15, the temperature was changed between 2.44 and 60.1°C at a frequency of 1.0 Hz and a strain (%) of 1%, and the changes in storage modulus and loss modulus were measured. As a result, the storage modulus decreased as the temperature increased, and the loss modulus increased after exceeding 35.5°C. At 60°C, the storage modulus and loss modulus values ​​became closer, and it is thought that if the temperature continues to increase beyond 60°C, the storage modulus will be less than the loss modulus. Furthermore, since the storage modulus was greater than the loss modulus at any temperature within the above range, it is thought that Sample 15 was a gel-like substance from 2.44 to 60.1°C, based on the results of the temperature dispersion test.

[0159] Viscoelastic Testing Considerations According to the results of the viscoelasticity test, among the bacterial preservative solutions, Samples 12 to 15, which are gel-like and miscible with feces, Sample 12, which has the lowest gellan gum concentration of 0.075% (w / v), had a linear region in the strain range of 3.98 to 39.8%, whereas Sample 15, which has the highest gellan gum concentration of 0.35% (w / v), had a linear region in the strain range of 0.0626 to 1.58%. Therefore, the linear region of bacterial preservative solutions with gellan gum concentrations between Samples 12 and 15 is thought to be between 0.063 and 39.8% in strain (%). Therefore, bacterial preservative solutions with strain (%) in this range are thought to be suitable bacterial preservative solutions that are gel-like and miscible with feces.

[0160] <Experiment 7: Preparation of gel-like bacterial preservative solution and growth of Bifidobacterium> Test Purpose To investigate the growth of Bifidobacterium in bacterial preservative solutions containing different amounts of gelling agent (complex polysaccharide (gellan gum)).

[0161] Test Method Bacterial preservative solutions with different gellan gum contents were prepared, and the growth state of Bifidobacterium in the mixture of the bacterial preservative solution and the fecal suspension was observed.

[0162] Preparation of bacterial stock solution: As in Experiment 6, five bacterial preservative solutions were prepared with different gellan gum concentrations and a pH of 4. The gellan gum concentrations and sample numbers of these five bacterial preservative solutions were as follows: Sample 17 with a gellan gum concentration of 0% (w / v), Sample 18 with a gellan gum concentration of 0.05% (w / v), Sample 12 with a gellan gum concentration of 0.075% (w / v) (same as in Experiment 6), Sample 19 with a gellan gum concentration of 0.175% (w / v), Sample 20 with a gellan gum concentration of 0.250% (w / v), and Sample 16 with a gellan gum concentration of 0.40% (w / v) (same as in Experiment 6).

[0163] Meanwhile, a fecal suspension was prepared by mixing approximately 2 g of feces collected from a human with 5 mL of sterilized water. 200 μL of this fecal suspension was then added to 5 mL of bacterial stock solution for each sample, mixed, and stored in a refrigerator at 4°C for one week. After one week, a portion was taken from this mixture, serially diluted in 1 / 10 increments, plated on agar plates, and anaerobically cultured at 37°C. The colonies formed on the plates were then counted to determine the number of viable Bifidobacterium bacteria in each mixture.

[0164] result The viable cell counts (cfu) of Bifidobacterium were 5.75E+06 for sample 17, 4.375E+06 for sample 18, 5.375E+06 for sample 12, 5.75E+06 for sample 19, and 5.75E+06 for sample 20. In addition, for sample 16, the bacterial preservative solution had hardened too much, and the addition and mixing of the fecal suspension did not go well, so subsequent storage and cultivation were not performed. From the above, the number of viable cells of Bifidobacterium did not change with the amount of gellan gum added, and gellan gum had no effect on the growth of Bifidobacterium. Furthermore, when the properties of the bacterial preservative solution for each sample were observed, sample 17 was a smooth liquid, while samples 18, 12, 19, and 20 were gel-like and easy to handle. As mentioned above, sample 16 became too hard and was difficult to handle. From the above, it was found that the gellan gum concentration should preferably be between 0.050 (w / v)% and 0.250 (w / v)%. [Explanation of symbols]

[0165] 10 Fecal collection kit 20 Inner container 21 Inner container body 22 Lid 23 Spoon Spatula 24 patterns 25 dent 26 Baffle Plate 27 Stirring ball 30 Outer container 31 Outer container body 32 Lid 33 Shielding membrane 40 Bacterial preservation solution

Claims

1. A method for manufacturing a product comprising a plurality of steps performed by a business operator, the plurality of steps comprising: a step of culturing a fecal collection solution containing feces collected from a human in an M2GSC medium and isolating Lactobacillus bacteria as useful bacteria derived from the human intestinal bacterial flora; A step of culturing the useful bacteria; storing the cultured beneficial bacteria; a step of obtaining a substance utilizing useful bacteria, which contains at least one of the stored useful bacteria, or a metabolic product or extract thereof; and providing the substance utilizing useful bacteria to the same person who collected the feces by the business operator. A method for using intestinal bacteria (excluding aspects of surgery, treatment, or diagnosis of humans).

2. Further comprising a bacterial flora analysis step of analyzing the feces collected from the human. A method for utilizing the intestinal bacteria described in claim 1.

3. The method further comprises a step of culturing the collected stool liquid on at least one of MGLP agar medium, modified BCP-added plate count agar medium, TOS mupirocin medium, TOS propionic acid agar medium, or paromomycin-containing TOS propionic acid agar medium, and isolating Bifidobacterium genus bacteria as useful bacteria derived from the human intestinal bacterial flora. A method for utilizing the intestinal bacteria described in claim 1.

4. The method further comprises a step of isolating at least one bacterium belonging to the genus Faecalibacterium, Roseburia, Akkermansia, Collinsella, Pediococcus, or Blautia as the useful bacterium. A method for utilizing the intestinal bacteria according to any one of claims 1 to 3.

5. A method for manufacturing a method for manufacturing a product, comprising: a step of culturing a fecal collection solution containing feces collected from a human in an M2GSC medium and isolating Lactobacillus bacteria as useful bacteria derived from the human intestinal bacterial flora; a step of obtaining a substance utilizing useful bacteria, which contains at least one of the useful bacteria and its metabolic products or extracts; and providing the substance utilizing useful bacteria to the same person who collected the feces by the business operator. A method for using intestinal bacteria (excluding aspects of surgery, treatment, or diagnosis of humans).

6. a step of culturing a fecal collection solution containing feces collected from a human in an M2GSC medium and isolating Lactobacillus bacteria as useful bacteria derived from the human intestinal bacterial flora; and obtaining a substance utilizing useful bacteria containing at least one of the useful bacteria, its metabolic products, or its extracts. A method for producing substances that utilize useful bacteria.

7. The method further comprises a step of culturing the collected stool liquid on at least one of MGLP agar medium, modified BCP-added plate count agar medium, TOS mupirocin medium, TOS propionic acid agar medium, or paromomycin-containing TOS propionic acid agar medium, and isolating Bifidobacterium genus bacteria as useful bacteria derived from the human intestinal bacterial flora. A method for producing a substance utilized by useful bacteria according to claim 6.

8. The method further comprises a step of isolating at least one bacterium belonging to the genus Faecalibacterium, Roseburia, Akkermansia, Collinsella, Pediococcus, or Blautia as the useful bacterium. A method for producing a substance utilized by useful bacteria according to claim 6.

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