Method for utilizing enterobacteria

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 an enteric bacteria bank. [Background technology]

[0002] Food taken in by humans is digested by digestive juices secreted from the digestive tract, and it is known that a huge number of microorganisms present in the intestines help digestion of food. These microorganisms are also called the intestinal flora, and not only help digest food, 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 this intestinal flora is related to various diseases such as obesity, allergies, Parkinson's disease, depression, and cancer. Therefore, attention has been drawn to the fact that the collection of microorganisms called the intestinal flora or intestinal microbiome plays an important role in maintaining human health.

[0003] For example, JP 2021-112218 A (Patent Document 1) describes an invention regarding 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. Also, for example, JP 2021-109865 A (Patent Document 2) describes an invention regarding an agent for improving the intestinal flora.

[0004] On the other hand, technologies 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] JP 2021-112218 A [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 type and amount of microorganisms present in the intestinal flora differs 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 differ from person to person. However, the conventional techniques described in the above Patent Documents 1 and 2 only focus on the general intestinal flora, and do not focus on the intestinal flora that differs from person to person. Therefore, there is a lack of a perspective of improving the intestinal flora in a custom-made manner according to the characteristics of each individual.

[0007] In addition, although the conventional technology described in Patent Document 3 above is capable of collecting microorganisms from human feces, there is no knowledge whatsoever regarding the use of the collected microorganisms, and no technology is disclosed that attempts to improve intestinal bacteria in a custom-made manner according to the characteristics of each individual.

[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 an intestinal bacteria bank business as an effort to achieve this. The intestinal bacteria bank business is one of the services that focuses on an individual's microbiome, and involves analyzing an individual's intestinal flora, collecting, multiplying, 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, the method 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 preservation solution and a stirring ball, and isolating useful bacteria from the feces collection solution that has been left for a predetermined period of time, 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 storage step of the stored useful bacteria, or a metabolic product or extract of the useful bacteria. and a useful bacteria utilization substance production step of obtaining a useful bacteria utilization substance containing at least one of the above, wherein the isolation step includes a useful bacteria isolation step of culturing the fecal collection liquid in any 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 fecal collection liquid in any 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 to isolate Bifidobacterium.

[0011] A first aspect of the present disclosure includes a bacterial flora analysis step of performing bacterial flora analysis on feces collected from a human, thereby making it possible to obtain information on what kind of bacteria the human has in his or her body through the bacterial flora analysis; an isolation step of mixing feces collected from the human into a feces collection kit containing a predetermined bacterial preservation 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 makes 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 makes 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 makes it possible to obtain the selected useful bacteria as desired. and a useful bacteria utilization substance production process 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 various beneficial products based on the useful bacteria. The isolation process includes a useful bacteria isolation process for culturing the fecal collection liquid in any of M2GSC medium, MRS medium, or MRS medium containing bile acid (cholic acid) to isolate Lactobacillus, and culturing the fecal collection liquid in any 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 to isolate Bifidobacterium, thereby making it possible to preferably isolate Lactobacillus and Bifidobacterium.

[0012] A second aspect of the present disclosure is a method for utilizing enterobacteria that utilizes useful bacteria in enterobacteria, comprising an isolation step of mixing feces collected from a human with a predetermined bacterial preservation solution and isolating useful bacteria from the fecal collection solution that has been left for a predetermined period of time, the isolation step comprising a step of culturing the fecal collection solution in any one 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 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 to isolate Bifidobacterium.

[0013] A second aspect of the present disclosure includes an isolation step of isolating useful bacteria from the fecal collection liquid that has been left for a predetermined period of time by mixing feces collected from a human with a predetermined bacterial preservation liquid, and thus useful bacteria contained in the human can be isolated and obtained. The isolation step includes a step of isolating useful bacteria by culturing the fecal collection liquid in any one of M2GSC medium, MRS medium, or MRS medium containing bile acid (cholic acid) to isolate Lactobacillus, and culturing the fecal collection liquid in any 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 to isolate Bifidobacterium, and thus Lactobacillus and Bifidobacterium can be suitably isolated.

[0014] A third aspect of the present disclosure is a method for utilizing enterobacteria that utilizes useful bacteria in enterobacteria, comprising an isolation step of mixing feces collected from a human with a specified bacterial preservation solution and isolating useful bacteria from the fecal collection solution that has been left for a specified period of time, the isolation step comprising culturing the fecal collection 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 a desired useful bacterium from all of the resulting colonies, thereby isolating the desired useful bacterium.

[0015] A third aspect of the present disclosure includes a step of culturing the fecal collection fluid on any one 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 the desired useful bacterium from all of the resulting colonies, thereby isolating the desired useful bacterium. Therefore, by using a medium in which multiple bacterial species can grow, the effort of culturing on multiple media can be eliminated.

[0016] A fourth aspect of the present disclosure is a method for using enterobacteria, 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 of 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 fecal collection solution in any one of M2GSC medium, MRS medium, or MRS medium containing bile acid (cholic acid) to isolate Lactobacillus, and culturing the fecal collection solution in any 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 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, comprising: mixing the feces with a bacterial preservative solution to obtain a fecal collection liquid; culturing the fecal collection liquid in any one of M2GSC medium, MRS medium, or MRS medium containing bile acid (cholic acid) to isolate Lactobacillus; and culturing the fecal collection liquid in any 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 to isolate Bifidobacterium, thereby enabling the 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 preservation solution to obtain a fecal collection solution, culturing the resulting 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 a desired useful bacterium from all of the resulting colonies to isolate the desired useful bacterium.

[0021] 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 preservation solution to obtain a fecal collection solution, culturing the resulting solution on either BCP 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 of the resulting colonies. Therefore, by using a medium in which multiple bacterial species can grow, the effort of culturing on multiple media can be eliminated.

[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 comprises isolating at least one bacterium from the genus 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, comprising: preparing a bacterial preservation solution in a buffer solution such as Dulbecco's phosphate buffered saline, which contains a reducing agent such as L-cysteine ​​hydrochloride and a cryoprotectant such as glycerol, and the pH of the solution is adjusted 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; culturing the collected fecal solution after a predetermined period of time in a medium such as M2GSC medium; and isolating useful bacteria such as Lactobacillus.

[0025] The other embodiment 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 with 5 to 10 mL of the bacterial preservation solution, culturing the collected feces after a predetermined period of time in a medium such as M2GSC medium, and isolating useful bacteria such as Lactobacillus, so that useful bacteria derived from an individual's intestinal flora can 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 preservation 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 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 total of the buffer solution and the cryoprotectant, so that the pH can be maintained within a predetermined range even after mixing with feces, making it a bacterial preservation solution suitable for retaining 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 preservation solution in which the buffer 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 kept within a predetermined range even after mixing with feces, making it a bacterial preservation solution suitable for retaining 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 preservation solutions.

[0033] The other embodiment is a stool collection kit comprising 5 to 10 mL of any of the bacterial preservation solutions described above, so that even if a desired amount of stool is collected and mixed with the bacterial preservation solution, the pH of the stool collection solution in this 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 specified period of time has passed.

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

[0035] The other embodiment is a method for isolating useful bacteria, in which a collected stool 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 produced the stool can be isolated from the stool.

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

[0037] The other embodiment is a method for isolating useful bacteria in which a stool sample is cultured in an M2GSC medium or the like to isolate useful bacteria such as Lactobacillus, and therefore it is possible to isolate necessary useful bacteria from the stool sample.

[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 preservation solution that has been contaminated with feces collected from the subject and allowed to sit 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 at least the cultured useful bacteria or a metabolic product or extract of the cultured useful bacteria.

[0039] The other aspect is a method for utilizing intestinal bacteria obtained from a subject, which includes the steps of isolating useful bacteria from a bacterial preservation solution that has been contaminated with feces collected from the subject and left for a predetermined period of time, freezing and preserving the isolated useful bacteria, culturing the frozen and preserved useful bacteria, and obtaining a content containing at least one of the cultured useful bacteria or a metabolic product or extract of the cultured useful bacteria, so that the useful bacteria actually held in the body of the subject can be utilized. Then, a content such as a capsule, supplement, or preparation containing a substance derived from the useful bacteria can be manufactured. Effect 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 utilized for producing products, etc. [Brief description of the drawings]

[0041]

Figure 1

Figure 2

Figure 3

[0042] One aspect of the present disclosure will be described with reference to the drawings based on an exemplary embodiment. The following embodiment does not unduly limit the contents of the present invention described in the claims. In addition, all of the configurations described in this embodiment are not 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. In addition, 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 different elements, they are not intended to indicate a specific order or superiority or inferiority. Configurations common to each embodiment are denoted by the same reference numerals and duplicated explanations are omitted.

[0046] The directions or positional relationships expressed by terms such as "upper", "lower", "front", "rear", "left", "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 there is a clear definition or limitation, it is not expressly or implied that a specific element or its use is configured in a specific direction, and it should be understood that it does not limit the scope of the claims and the embodiments.

[0047] Numerical values ​​or elements modified by the terms "about," "approximately," "nearly," and "substantially" used in this specification and claims are understood to include the numerical value and the numerical values ​​before and after the numerical value, and to include the element and those that can be said to be the same as the element. For example, when describing "about 3," "3" and the numerical values ​​following it can be included in "about 3" as long as the technical features and technical meanings claimed in this disclosure are not different. In addition, when describing "B that is substantially the same as A," B can be included in the range of "substantially" even if it has differences, as long as the technical features and technical meanings claimed in this disclosure are common.

[0048] The symbol "~" used in this specification and claims is understood to include the lower limit and the upper limit, and also includes any value between the lower limit and the upper limit, unless otherwise specified. For example, "pH 3.5-5" means "pH 3.5 or more and 5 or less," and "0.005-0.02 g" means "0.005 g or more and 0.02 g or less."

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

[0050] 1. Bacterial flora analysis There are bacteria that live in the human body all the time, called resident human flora. These bacteria are diverse and differ depending on each organ and tissue of the human body. For example, there are resident skin bacteria that adapt to the environment exposed to the outside world, and anaerobic bacteria exist in digestive organs such as the intestines, where oxygen is difficult to reach. The conventional approach to these resident human flora has been to study bacteria such as pathogenic bacteria that cause illness and deterioration of health in the human body, and to eliminate or suppress these bacteria. However, elucidating only the pathogenic bacteria that cause harm to the human body has not been able to contribute to the true health of the human body. For example, in today's world, where it is too late to become 100 years old after getting sick, the goal has become to live a long and healthy life, not to live a long life. When people are expected to stay healthy all the time without getting sick, it is not enough to just study and take measures against pathogenic bacteria.

[0051] In recent years, research in the field of genetics has progressed and analytical techniques have improved. For example, the advent of next-generation sequencers has made it possible to analyze bacterial flora, which was previously difficult. Using this technology that uses next-generation sequencers, it is now possible to analyze bacteria that have not been identified before because they are difficult to culture. From another perspective, it is now possible to analyze bacterial flora that includes not only bacteria that have adverse effects on 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 become clear that visualizing bacterial flora that includes such various bacteria can be very useful in 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, the company has 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 considered necessary to first analyze the intestinal flora of a specific individual. For this reason, it is preferable to collect feces from an individual and analyze the flora contained in the feces. We decided to analyze human feces to analyze the intestinal bacterial flora because intestinal bacteria, both live and dead, are excreted from the body in feces, making them easy to collect without using surgical procedures on the human body.

[0054] It is preferable to use the above-mentioned next-generation sequencer for the analysis of intestinal microbiota. This is because the next-generation sequencer determines the gene sequences in DNA or RNA samples of multiple types of bacteria simultaneously, and classifies and identifies bacteria based on a base sequence database that stores the base sequence of the bacterial gene, its description, and literature information. Since the conventional Sanger method can only analyze one gene sequence of one type of bacteria, the use of the next-generation sequencer is superior for analyzing the 16SrRNA gene of the intestinal microbiota, which is packed with a huge amount of genetic information.

[0055] In the analysis by next-generation sequencers, the mainstream approach is to classify the intestinal bacteria in feces by "phylum," "genus," and "species." When classifying by "phylum," "genus" and "species" are aggregated into one group, so if only the "phylum" notation is used, it is difficult to distinguish the characteristics of individual bacterial species and strains. In addition, when classifying by "species," it is possible to distinguish the characteristics of individual bacteria, but the classification rate is low, and analysis of the 16SrRNA gene by next-generation sequencers lacks accuracy in classification. Classification by "genus" allows the characteristics of individual bacteria to be distinguished compared to "phylum," and it is possible to classify with a higher classification rate than "species." For these reasons, the "genus" level is preferable when classifying the intestinal bacterial flora.

[0056] The analysis results are obtained as the names of the classified bacteria and their numbers or occupancy rates, but there is no useful value in randomly listing them. 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 order of the number of bacteria contained in feces, along with their characteristics and functions, because it is believed that the effects of the bacteria in the top ranks are strongly reflected in the human body.

[0057] Another output format is to show a list of bacteria contained in feces that are considered to be beneficial and a list of bacteria that are considered to be harmful, along with their characteristics and functionality. This is because it allows the user to know the types of beneficial and harmful bacteria that the individual possesses, and the names of those bacteria. Still another output mode is to assign the types and amounts of bacteria present to several categories with predefined evaluations, and to show 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 method 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 are most prevalent in 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, keep the intestinal pH low, and suppress bacteria such as Escherichia coli to regulate the intestinal environment. Type F is a type that is rich in Faecalibacterium bacteria, which are said to produce butyric acid and contribute to disease prevention and improving bowel movements. Type R is a type that is rich in Ruminococcus bacteria, which are said to break down water-soluble dietary fiber and produce short-chain fatty acids related to the immune system. Type P is a type that is rich in Prevotella bacteria, which break down dietary fiber and produce succinic acid and acetic acid.

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

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

[0062] 3.Explanation of how to utilize 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 with reference to 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 is stored in a designated feces collection kit containing a designated bacterial preservation solution and delivered to an institution for isolating and culturing the bacteria.

[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 performing bacterial flora analysis 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 left in the fecal collection kit for a specified period of time (ST3 in Figure 1). Since there are various species of bacteria in feces, including not only good bacteria but also bad 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 among the same cultured species (ST5 in Figure 1). This is to collect the best strains by taking into consideration the ease of growth, resistance, etc.

[0070] (6) Preservation of selected bacteria (storage process) This is the process of preserving the desired selected bacteria (ST6 in Figure 1). The selected bacteria are mixed with a preservation 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 multiplied for various uses. Various substances utilizing useful bacteria are produced, such as the useful bacteria themselves or extracts from the useful bacteria, capsules, tablets, and other supplements containing metabolic products of the useful bacteria. The substances utilizing useful bacteria are then provided to the individual from whom the useful bacteria were obtained, or to others who wish to multiply the 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 the intestinal flora of an individual are identified from the analysis based on the flora analysis, and the information is provided and utilized. For example, by analyzing the initial analysis based on the flora analysis and the changes in the intestinal flora after a change in diet or the intake of supplements, the effects of changes in diet or supplements can be verified.

[0073] The method for utilizing enterobacteria (enterobacteria bank) according to one embodiment of the present disclosure is carried out as described above. However, the 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 replaced if there is no technical inconsistency.

[0074] Furthermore, the method of using intestinal bacteria (intestinal bacteria bank) can be configured to be performed by an individual only once, or can be configured to be performed multiple times over time. When an individual continuously performs the method of using 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 bacteria 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 enterobacteria

[0076] Examples of embodiments of various technologies that can be used to realize the above-mentioned method for utilizing enterobacteria (enterobacteria 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 an individual (human) without going through a surgical procedure, it is preferable to collect the bacteria 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 to collect 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 preservation solution in a specified container.

[0079] Bacterial preservation solution: First, the bacterial preservation liquid enclosed in the stool collection kit must preserve the bacterial flora contained in the stool for a predetermined period of time until the next step is taken, without killing the bacteria. The "predetermined period" is the period from when the stool is placed in the stool collection kit until the isolation of useful bacteria is started at the testing institution, and specifically, it is a period of 1 day to 1 month, preferably 2 days to 2 weeks, more preferably 10 days to 14 days, and even more preferably a period of 1 week or about 1 week. The reason for this is that it takes several days to transport the collected stool to the testing institution, and even if the stool collection kit containing the collected stool arrives at the testing institution, it may take several days to collect and culture the microorganisms. From this perspective, the following bacterial preservation liquid 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, and the optimal pH is 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 has the function of suppressing the influence of pH. Specific examples of the buffer solution include, but are not limited to, phosphate buffer solution, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer solution, Tris buffer solution, carbonate buffer solution, bicarbonate buffer solution, acetate buffer solution, citrate buffer solution, phosphate buffered saline (PBS), HEPES buffered saline, Tris buffered saline, and the like. 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 called "Dulbecco's phosphate buffered saline solution") not containing divalent cations. The reason why Dulbecco's phosphate buffered saline is more preferable is that it minimizes the pH fluctuation of the bacterial preservation solution caused by mixing with feces, and can keep the bacteria in the feces alive without damaging them. The "Dulbecco's phosphate buffered saline, etc." described 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, methylene blue, etc. L-cysteine ​​hydrochloride is used as a food additive and is also used to reduce oxygen in water to create anaerobic conditions.

[0083] The pH adjuster is used to reduce stress on bacteria in feces by adjusting the strongly acidic (pH 2 or more and less than 3) bacterial preservation solution to a weakly acidic (pH 3 or more and less than 6, preferably pH 3.5 to 5) pH adjuster. Examples of pH adjusters include, but are not limited to, carbonates, bicarbonates, hydrochloric acid, sodium hydroxide, and the like. More specifically, the pH adjuster is preferably sodium hydrogen carbonate or sodium carbonate, and among these, sodium hydrogen carbonate is more preferable. This is because sodium hydrogen carbonate is suitable for the growth of methane bacteria, which are anaerobic bacteria, and is easy to finely adjust the pH, and sodium hydrogen carbonate is generally used as baking soda, and is therefore highly safe, while hydrochloric acid and sodium hydroxide are toxic and deleterious substances. The term "sodium hydrogen carbonate, etc." in the claims may include the various pH adjusters exemplified above.

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

[0085] It is preferable to prepare the bacterial preservation solution so that its pH is 3.5 or more and 5 or less. This is because useful bacteria can survive if the pH of the stool collection solution after a required amount of stool is placed in a stool collection kit and mixed with the bacterial preservation solution is 5 or more, and therefore the pH of the bacterial preservation solution may be 4 to 5, or 3.5 to 5, in order to achieve a pH of 5 or more 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 and lower than 3), which is a significant stressor for the intestinal bacteria; however, in order to reduce stress on the intestinal bacteria while maintaining the effect of the reducing agent, the solution is adjusted to a weak acidity (pH 3 or higher and 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 the total of the buffer solution and cryoprotectant. If the amount is less than 0.005 g, there is no reducing effect, and if the amount is more than 0.02 g, the pH becomes too low.

[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 of the buffer solution and the cryoprotectant. If the content is less than 0.5 mg, it is difficult to adjust the pH, and if the content is less than 5 mg, it may not be possible to adjust the pH. If the content exceeds 2 mg, 10 mg, or 30 mg, the effects of other ingredients may be weakened.

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

[0090] A preferred embodiment of the bacterial preservation 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 typical feces preservation solution, useful anaerobic bacteria often die, but the above-mentioned bacterial preservation solution is suitable for preserving anaerobic bacteria and reduces the possibility of useful bacteria dying 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 enteric bacteria bank.

[0092] In another embodiment of the bacterial preservation solution, said bacterial preservation solution further comprises a gelling agent. This bacterial preservation solution has the property that it becomes a non-flowing gel when left for a specified period of time, and becomes a flowable liquid when mixed with feces and shaken. "A gel that does not flow when left for a specified time" means that it becomes a gel that loses fluidity when left at room temperature for at least one day, and changes to a fluid sol when shaken. When mixed with feces and shaken, the bacterial preservation solution and feces mix to become a fecal collection liquid, and this fecal collection liquid is in a fluid sol state. The fecal collection liquid is in a state of mixed feces, and when stored 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) relative to the bacterial preservative solution, and more preferably 0.075 to 0.25% (w / v). The reason for setting the range as above is that if the amount is less than 0.05% (w / v), the gelling agent is in a liquid state and the effect of the gelling agent cannot be obtained, and if the amount is more than 0.35% (w / v), it becomes difficult to mix the feces uniformly with the bacterial preservative solution. On the other hand, in 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, and the handling and operability of the feces collection kit equipped with the bacterial preservative solution are improved. In addition, in the range of 0.075 to 0.25% (w / v), the growth of bifidobacteria is also excellent.

[0094] Gel-type bacterial preservation solutions are superior in preserving bacteria that are difficult to culture compared to liquid bacterial preservation solutions. In other words, feces contains fewer bacteria in the first place than bacteria that are easy to culture, such as Bifidobacterium, and it may be possible to culture certain types of bacteria that are difficult to isolate and culture later. Although the reason for this is unclear, it is speculated that the change in the bacteria when exposed to the environment from feces to the bacterial preservation solution is gentler because the bacterial preservation solution is gel-type rather than liquid-type. In addition, it is thought that the inclusion of a gelling agent disperses and stabilizes the buffer, reducing agent, cryoprotectant, and pH adjuster contained in the bacterial preservation solution, thereby improving the preservation of the bacteria.

[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 produced by the process known as gelling from Pseudomonas elodea ( Pseudomonas elodea ) after deacetylation and purification, 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 may inhibit the swarming of some microorganisms, which may hinder the culture. The reason why gellan gum is suitable for culturing microorganisms that are difficult to culture is that the bacterial preservation solution has an appropriate viscosity and therefore acts gently on the environmental changes of the bacteria, and it is thought that gellan gum has a supporting effect on the difficult-to-culture bacteria by acting on things other than the bacterial body, such as extracellular enzymes related to the uptake of nutrients.

[0096] container: The stool collection kit consists of a container containing a bacterial preservative solution.

[0097] The container is preferably composed of 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 preservation liquid. The lid is for sealing the opening of the container body, and is preferably provided with a spoon-shaped spatula on the inside of the lid for scraping off feces and placing it in the container body. The spatula is preferably provided with a recess in which about 1 to 2 g of feces can be placed. This is because the desired amount of 1 to 2 g of feces can be collected by scraping off feces with the tip of the spatula and placing the feces on the tip. However, since a fairly large recess is required to place 1 to 2 g of feces, 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 feces, so that feces can be collected two or three times.

[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 bacteria preservation liquid 40, and a lid 22 for sealing the bacteria preservation liquid 40 in the inner container body 21. A spoon-shaped spatula 23 attached to the lid 22 has a recess 25 on the tip of a handle 24 for placing feces thereon, and a baffle plate 26 near the recess 25 of the handle 24 for suitably mixing the feces into the bacteria preservation liquid 40. The recess 25 may be bent from the handle 24, or may be straight extending 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. Since the inner container 20 is placed inside the outer container 30, even if the fecal collection liquid in which feces is mixed with the bacteria preservation liquid 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 out. For the inner container 20 and the outer container 30, commercially available containers manufactured by Sarstedt or the like can be used.

[0101] It is preferable to put a predetermined amount of stirring balls 27 in the inner container 20. Since the bacteria preservation liquid 40 becomes gel-like and has no fluidity when left for a while, it is preferable to shake the inner container 20 to mix the feces after putting it in, but if the stirring balls 27 are in the inner container 20, it helps to fluidize the bacteria preservation liquid, making it easier to mix the bacteria preservation liquid with the feces. The stirring balls 27 may be any suitable ball that contributes to mixing the bacterial preservation solution with the feces and promotes mixing of the feces and the bacterial preservation solution, but are preferably stainless steel balls with a diameter of 2 to 4 mm, 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 by placing 3 to 10 balls with a diameter of 2 to 4 mm, the stirring efficiency can be improved. If the balls are smaller than 2 mm, larger than 4 mm, or less than 3, the stirring efficiency will be poor. Also, if balls larger than 4 mm or more than 10 balls are placed, the amount of the bacterial preservation solution 40 will be relatively small, and this will lead to increased costs, which is not preferable. One embodiment of using the 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 useful bacteria may not be collected. Furthermore, since the necessary useful bacteria can be collected with an amount up to 3 g, collecting feces in excess of 3 g increases the amount of bacterial preservation solution required and requires a larger inner container, resulting in high costs. The amount of bacterial preservation 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 is too small for the amount of stool to be collected, and there is a risk that useful bacteria in the stool will die. If it exceeds 20 mL, the inner container will become larger and the cost will increase.

[0103] The inner container preferably contains 30 to 90 vol% of the bacterial preservation solution. If the content is less than 30 vol%, the protection of the bacteria in the feces may be insufficient, and the amount of voids that may be mixed in when the feces is mixed into the inner container may increase, which may impair the survival of anaerobic bacteria. If the content exceeds 90 vol%, the feces collection solution may overflow from the inner container when the feces is 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 collected amount of feces can be placed in the inner container containing the above proportion of bacterial preservation solution and the inner container can be covered.

[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 preservation solution. This is because the stool may not be fully enveloped in the bacterial preservation solution and may come into contact with air, causing the beneficial bacteria to die. 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 for isolating the bacteria. It is preferable to store the stool collection kit in a refrigerator between collection and delivery. Since it is desirable to collect bacteria from the stool as soon as possible, it is preferable to send the kit refrigerated for 2 days or less after collection.

[0106] <Isolation process of useful bacteria> This is a process in which specific beneficial bacteria are isolated from the many types of bacteria contained in feces.

[0107] Beneficial bacteria are bacteria that have a beneficial effect 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 amounts in feces depending on the individual. Therefore, it is effective to isolate Lactobacillus from individuals whose bacterial flora has been analyzed by next-generation sequencing (NGS) and confirmed to have Lactobacillus.

[0108] Other useful bacteria include the genus Faecalis bacterium ( Faecalibacterium ) and Roseburia spp. Roseburia ) and other butyric acid-producing bacteria such as 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 sample that corresponds to the bacteria to be isolated. This is because it is easier to isolate the desired bacteria if the growth of bacteria other than the desired bacteria can be eliminated. Examples of such media include media in which selective agents such as antibiotics that inhibit the growth of other bacteria are added to media in which bacteria other than the target bacteria can also grow, 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 the properties unique to the desired bacteria.

[0110] When isolating Bifidobacterium as a specific liquid medium according to the bacterium, examples of the liquid medium include MGLP agar medium, modified BCP-added 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 contamination with lactic acid bacteria that tend to coexist with bifidobacteria.

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

[0112] When isolating Lactobacillus, 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 intestine can be selectively obtained.

[0113] Alternatively, instead of using a selective medium that does not allow the growth of bacteria other than a specific useful bacteria, a medium in which various bacteria can grow can be used for cultivation, and then the desired useful bacteria can be isolated by extracting DNA from a colony having the characteristics of the desired useful bacteria from among all the resulting colonies, identifying the bacterial species, etc. 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-added 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 sensitive to oxygen, 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 (bromocresol purple) contained in the medium turns yellow when lactic acid bacteria produce lactic acid. In calcium carbonate-containing MRS agar medium, the medium itself is cloudy due to the calcium carbonate contained therein, but since lactic acid bacteria dissolve the calcium carbonate, the area around the lactic acid bacterial colonies becomes transparent, making it easier to detect the lactic acid bacteria.

[0115] After culturing in these media, DNA is extracted from the resulting colonies to identify the bacterial species, allowing the desired useful bacteria to be isolated.

[0116] <Isolated Bacteria Selection Process> 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 bile acid tolerance test is performed on multiple isolated strains to find a strain with high resistance to bile acids, it is believed that the bacteria will also have high resistance to gastric acid and will be more likely to reach the intestine. In this way, various selection steps such as the bile acid tolerance test can be added to obtain bacteria with stronger preferred properties.

[0117] <Preservation (storage) process of selected bacteria (process of enriching isolated bacteria)> The isolated and identified bacteria can be mixed with a preservative solution along with the culture medium, placed in a designated tube, and frozen for storage to preserve the 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 tube having a capacity of 1.5 to 2.0 mL is preferable.

[0118] Various media such as MRS medium can be used as the culture solution containing useful bacteria before mixing with the cryoprotectant, and it is preferable that the medium contains the isolated useful bacteria at a content of 1.00E+08 / mL or more, or at a point where the growth curve reaches the stationary phase. This is because it is thought that cells in the stationary phase are more resistant to freezing stress than cells in the logarithmic growth phase, and since a certain percentage of cells die due to freezing, if the content is less than 1.00E+08 / mL, there is a risk that the cells will not grow to the required amount when used, and the preservation efficiency of the useful bacteria is poor. Furthermore, it is practically difficult to contain a content significantly exceeding 1.00E+08 / mL.

[0119] The content of the cryoprotectant in the mixture obtained by mixing the culture solution containing the useful bacteria with a cryoprotectant is 15 to 20 vol%. If the proportion of the cryoprotectant is less than 15 vol%, the culture solution may freeze during freezing, and the useful bacteria may be destroyed. On the other hand, if the proportion of the cryoprotectant is more than 20 vol%, the proportion of the useful bacteria contained in the liquid decreases, making it difficult to grow these useful bacteria when they are used, and the preservation efficiency is poor.

[0120] After mixing the medium containing useful bacteria with a cryoprotectant, the obtained storage liquid can be stored in a tube under the following conditions: storage at -196°C to -160°C using liquid nitrogen, or storage in an ultra-low temperature freezer with a temperature of -150°C, -90°C to -80°C, -60°C to -40°C, or -35°C to -20°C. The lower the temperature, the more advantageous it is that the metabolic activity of the bacteria can be stopped and the dormant state of the cells can be maintained for a long period of time, but at -196°C to -150°C, the costs associated with maintaining cooling and the need to secure a wide area are required. In addition, at -60°C to -20°C, the sample is easily affected by the surrounding temperature, and the temperature inside the storage cabinet fluctuates greatly when the storage cabinet is opened and closed, which leads to deterioration of the quality of the frozen samples, making it difficult to store the samples for a long period of time. Therefore, it is preferable to store the samples frozen at around -80°C using an ultra-low temperature freezer with a temperature of -90°C to -80°C.

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

[0122] Alternatively, by providing the above-mentioned capsules, supplements, etc. derived from the banked specific useful bacteria to an individual who does not have the specific useful bacteria or who has them in small numbers, it is possible to contribute to improving the intestinal flora of that individual. EXAMPLES

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

[0124] Preparation of bacterial stock solution: D-PBS(-), glycerin solution, L-cysteine ​​hydrochloride, and sodium bicarbonate solution were mixed to prepare six types of bacterial preservation solutions with D-PBS(-) concentrations of 50% (v / v), L-cysteine ​​hydrochloride concentrations of 0.1% (w / v), and glycerin concentrations of 25% (v / v), and pH values ​​of 3.0, 3.5, 4.0, 4.5, 5.0, and 5.5, which were designated as samples 2 to 7. In addition, a bacterial preservation solution with no added sodium bicarbonate and no pH adjustment (NC) was also prepared and designated as sample 1. The pH of this unadjusted bacterial preservation solution was strongly acidic, ranging from 2.7 to 2.8.

[0125] Test Method: Approximately equal amounts of feces from the same person defecated at the same time were added to 10 mL of the above sample, and suspended completely until all fecal masses disappeared. -3 , 10 -4 , 10 -5 The final dilution was applied to TOS medium, a plate medium dedicated to Bifidobacterium, and cultured for 72 hours under anaerobic conditions at 37°C. After culture, the number of colonies formed on the plate medium 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. In addition, compared to sample 1, whose pH was not adjusted, and sample 7, whose pH was 5.5, the results of the four viability runs for these samples were concentrated in the middle.

[0127] On the other hand, in sample 1, where the pH was not adjusted, the survival rate was sometimes 0, and the median was the furthest from the standard value of 1.0. Sample 7, with 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 preservation solution with a pH adjusted to 3.5 to 5, it is possible to keep Bifidobacterium in the fecal fluid for a specified period of time without killing them.

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

[0130] Test Method We attempted to isolate Lactobacillus using M2GSC medium and MRS medium containing bile acid (cholic acid). The same fecal collected solution mixed with feces used in Experiment 1 (pH 4.5 after preparation of bacterial preservation solution, pH 6.39 after mixing with feces, sample 5a) 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 for 72 hours under anaerobic conditions.

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

[0132] Test results: All three media produced round yellow or round white colonies, but the number of round yellow colonies was smaller in the normal MRS medium than in the M2GSC medium and the MRS medium containing bile acids.

[0133] All of the bacteria obtained from the six colonies selected from M2GSC medium and standard MRS medium showed high homology (98% or more) with Lactobacillus species. However, the species obtained were biased depending on the medium, and only Lacticaceae Bacillus paracasei ( Lacticaseibacillus paracasei ) or Lactiprunci Bacillus plantarum ( Lactiplantibacillus plantarum ) preferentially grew in the normal MRS medium, whereas Rimocilactobacillus fermentum ( Limosilactobacillus fermentum ) was found to grow preferentially.

[0134] Needle-shaped crystals were confirmed 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 cholesterol in the blood, 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 ), Lentilactobacillus senior ( Lentilactobacillus senioris ), Lactipranchi Bacillus fabifermentans ( Lactiplantibacillus fabifermentans ) and other lactic acid bacteria were also identified, with Lactiplantibacillus 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, a TOS agar medium was autoclaved (115° C., 15 minutes) and then cooled to 50° C., and 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 matter 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 smeared on 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 base sequence of the amplified PCR product was analyzed by a sequencer, and the bacterial species was identified using the NCBI Blast search system.

[0139] Test results: The bacteria identified from the single colonies obtained 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 from feces.

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

[0142] <Experiment 5: Functional evaluation of bacterial preservation 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 "conventional preservation solution") is described in Patent Document 3 (US Patent Application Publication No. 2022 / 0160337). However, since the pH of this conventional preservation solution is 7.5, there are doubts about the preservation of anaerobic bacteria in feces. Therefore, the preservation performance of Akkermansia, which is an intestinal bacterium, a useful bacterium, and an obligate anaerobic bacterium, 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 composition of the bacterial preservation solution of the present invention was as follows and designated Sample 8. Bacterial preservation solution of the present invention (composition): · L-cysteine ​​hydrochloride……1g / L 50% (w / v) glycerol……500mL D-PBS……500mL ·NaHCO3……Add as needed (to pH 4.0)

[0146] On the other hand, the conventional preservation solution was the one described in the specification, Table 1 of Patent Document 3. The composition of the solution was as follows, and this solution was designated as 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 preservation solution having the above composition was 7.5.

[0147] (3) After culturing the Akkermansia, the supernatant and the cell pellet of Akkermansia 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 -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 under anaerobic conditions for 72 to 96 hours. After culture, the colonies formed on the plates were counted, and the viable cell count of Akkermansia in the suspension was calculated.

[0148] (4) 500 μL of the Akkermansia suspension prepared in (3) above was added to and mixed with 5 mL of each of Sample 8, the bacterial preservation solution of the present invention prepared in (2) above, and Sample 9, the conventional preservation solution, and stored at 4° C. for 5, 7, and 10 days. After storage, these mixtures were -3 , 10 -4 80 μL of each dilution was added and spread onto modified GAM plate medium and cultured under anaerobic conditions at 37°C for 72 to 96 hours. After culture, the colonies formed on the plate medium were counted and the number of live 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 defined as the survival rate of Akkermansia for each day that has passed. That is, the survival rate (%) = number of viable bacteria cfu after storage at 4°C (5, 7, 10 days) × 9.09 / number of viable bacteria cfu in 500 μL Akkermansia suspension × 100. Note that 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 "0.5 mL / 5.5 mL × 100 = 9.09 times."

[0150] result The viable cell counts (cfu) of sample 8, the bacterial preservation solution of the present invention, and sample 9, the conventional preservation 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% (0 days), 57.4% (5 days), 20.6% (10 days) Sample 9: 100% (0 days), 16.5% (5 days), 8.9% (10 days)

[0151] Consideration From the results of the survival rate of Akkermansia, the bacterial preservation solution of the present invention (sample 8) had a higher viable cell count and survival rate of Akkermansia than the conventional preservation solution (sample 9). The reason for this is considered to be that the bacterial preservation solution of the present invention is adjusted to an acidic pH of 4 to enhance the deoxidizing effect of the reducing agent L-cysteine ​​hydrochloride, whereas the pH of conventional preservation solutions is adjusted to a weakly alkaline pH of 7.5, so that the reducing effect of L-cysteine ​​hydrochloride could not be maintained in the preservation solution. In addition, the bacterial preservation solution of the present invention uses NaHCO3 when adjusting the pH, which is thought to have a synergistic effect on the deoxidizing effect with L-cysteine ​​hydrochloride.

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

[0153] <Experiment 6: Preparation of gel-like bacterial preservation solution and viscoelasticity measurement> Preparation of bacterial stock solution: D-PBS(-), glycerin solution, L-cysteine ​​hydrochloride, gellan gum, and sodium bicarbonate solution were mixed. More 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 was mixed with the glycerin solution with gellan gum dissolved therein at a 1:1 ratio (vol). Six types of bacterial preservation solutions were prepared with a D-PBS(-) concentration of 50% (v / v), an L-cysteine ​​hydrochloride concentration of 0.1% (w / v), a glycerin concentration of 25% (v / v), and different gellan gum concentrations, with a pH of 4. The gellan gum concentrations and sample numbers of these six bacterial preservation solutions are as follows: The gellan gum concentrations are sample 11 of 0.02% (w / v), sample 12 of 0.075% (w / v), sample 13 of 0.1% (w / v), sample 14 of 0.3% (w / v), sample 15 of 0.35% (w / v), and sample 16 of 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 when gellan gum was not added, but was liquid and did not gel. On the other hand, Samples 12 to 16 were gel-like and did not flow or flow even when the tube containing the sample was tilted. Furthermore, when feces was added to the bacterial preservation solutions of Samples 12 to 15, the feces mixed with the bacterial preservation solution, whereas when feces was added to the bacterial preservation solution of Sample 16, the feces did not mix completely with the bacterial preservation 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 in the range of strain (%) from 0 to 1000 at 30°C and 1.0 Hz) 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 between strains of 3.98% and 39.8%, there was little change in both moduli and they were in the stable linear region. On the other hand, for sample 15, with 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 changes in both moduli were small between strains of 0.0626% and 1.58%, and they were in the stable linear region.

[0156] Viscoelasticity measurement (2)……Frequency dispersion For the above-mentioned Samples 12 and 15, strains (%) were 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. 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 substance based on the results of the frequency dispersion test.

[0157] Viscoelasticity measurement (3)...Temperature dispersion For the above-mentioned samples 12 and 15, strains (%) were 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 change in storage modulus and loss modulus was measured by changing the temperature between 2.55 and 60° C. with a frequency of 1.0 Hz and a strain (%) of 10%. As a result, the storage modulus decreased with increasing temperature, and the material showed a tendency to progress to a liquid (sol). In addition, 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 material from 2.55 to 60°C, based on the results of the temperature dispersion test.

[0158] For Sample 15, the changes in storage modulus and loss modulus were measured by changing the temperature between 2.44 and 60.1° C. with a frequency of 1.0 Hz and a strain (%) of 1%. As a result, as the temperature increased, the storage modulus decreased, and the loss modulus increased after exceeding 35.5°C. At 60°C, the values ​​of the storage modulus and the loss modulus became close to each other, so it is considered that if the temperature continues to increase beyond 60°C, the storage modulus will be less than the loss modulus. In addition, since the storage modulus was greater than the loss modulus at any temperature within the above range, it is considered that sample 15 is 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 preservation solutions of Samples 12 to 15, which are gel-like and mix 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, it is considered that the linear region of bacterial preservation solutions with gellan gum concentrations between Samples 12 and 15 is between 0.063 to 39.8% in strain (%). Therefore, it is considered that bacterial preservation solutions with strain (%) in this range are suitable bacterial preservation solutions that are gel-like and mix with feces.

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

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

[0162] Preparation of bacterial stock solution: As in Experiment 6 above, five types of bacterial preservation solutions were prepared with different gellan gum concentrations and a pH of 4. The gellan gum concentrations and sample numbers of these five bacterial preservation solutions are 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 human feces with 5 mL of sterilized water. Then, 200 μL of this fecal suspension was added to 5 mL of bacterial stock solution of each sample, mixed, and stored at 4°C in a refrigerator for one week. After one week, a portion was taken from this mixture, serially diluted in increments of 1 / 10, spread on a plate medium, and cultured anaerobically at 37°C. The colonies formed on the plate medium were then counted to calculate the number of viable Bifidobacterium bacteria in each mixture.

[0164] result The viable cell counts of Bifidobacterium (cfu) 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 became too hard, and the addition and mixing of the fecal suspension did not go well, so subsequent storage and cultivation were not performed. From the above, there was no change in the viable cell count of Bifidobacterium due to changes in the amount of gellan gum added, and gellan gum had no effect on the growth of Bifidobacterium. Furthermore, when the properties of the bacterial preservation solution of each sample were observed, sample 17 was a smooth liquid, whereas 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 step of culturing a fecal collection liquid containing feces collected from a human in an M2GSC medium and isolating Lactobacillus bacteria as useful bacteria derived from the human intestinal flora; A step of culturing the useful bacteria; storing the cultured useful bacteria; A step of obtaining a useful bacterial utilization substance containing the stored useful bacteria or at least one of a metabolic product or extract thereof; Providing the useful bacteria-utilized substance to the human individual from whom the feces was collected. A method for using enterobacteria (excluding aspects of surgery, treatment, or diagnosis of humans).

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

3. The method further comprises a step of culturing the fecal collection 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 a Bifidobacterium genus bacterium as a useful bacterium derived from the human intestinal flora. A method for utilizing the intestinal bacteria described in claim 1.

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

5. A step of culturing a fecal collection liquid containing feces collected from a human in an M2GSC medium and isolating Lactobacillus bacteria as useful bacteria derived from the human intestinal flora; A step of obtaining a useful bacterial utilization substance containing the useful bacteria or at least one of a metabolic product or an extract thereof; Providing the useful bacteria-utilized substance to the human individual from whom the feces was collected. A method for using enterobacteria (excluding aspects of surgery, treatment, or diagnosis of humans).

6. A step of culturing a fecal collection liquid containing feces collected from a human in an M2GSC medium and isolating Lactobacillus bacteria as useful bacteria derived from the human intestinal flora; Obtaining a useful bacterial utilization substance containing at least one of the useful bacteria or a metabolic product or extract thereof. A method for producing substances utilizing useful bacteria.

7. The method further comprises a step of culturing the fecal collection 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 a Bifidobacterium genus bacterium as a useful bacterium derived from the human intestinal flora. A method for producing a substance utilized by useful bacteria according to claim 6.

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

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