Method for preserving intestinal bacteria, bacterial preservation solution, and fecal collection kit

Abstract

To provide a method of using intestinal bacteria, a bacterial preservation solution and a stool sampling kit which are related to a method for sampling and using useful bacteria contained in stool excreted by an individual person.SOLUTION: A method of using intestinal bacteria that uses beneficial bacteria in intestinal bacteria, includes: a bacterial flora analyzing step of analyzing bacterial flora of stool sampled from a human; an isolating step of isolating the beneficial bacteria from the stool sampled liquid after a predetermined period of time elapses since mixing the stool sampled from the human with a stool sampling kit in which a predetermined bacterial preservation solution and agitation balls are contained; a cultivating step of cultivating the isolated beneficial bacteria; a selecting step of selecting excellent beneficial bacteria from among the beneficial bacteria acquired in the cultivating step; a storing step of storing the selected beneficial bacteria; and a substance-using-beneficial-bacteria producing step of acquiring and producing a substance using the beneficial bacteria, containing at least any one of the stored beneficial bacteria, metabolite thereof, or extract. The bacteria preservation solution, which is prepared with a specific reagent, becomes gelatinous without flowing when left untouched for a specified period of time.SELECTED DRAWING: Figure 2

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] U.S. Patent Application Publication No. 2022 / 0160337

SUMMARY OF THE INVENTION

PROBLEMS TO BE SOLVED BY THE INVENTION

[0006] Briefly speaking, although it is a group of bacteria called the gut microbiota, the gut microbiota varies from person to person in terms of the types and amounts of microorganisms present, and the types and amounts of so-called beneficial bacteria considered beneficial to the body and harmful bacteria considered harmful to the body also differ from person to person. However, the prior art described in Patent Document 1 or Patent Document 2 focuses only on the general gut microbiota and does not focus on the gut microbiota that varies from person to person. Therefore, it lacked the perspective of improving the gut microbiota in a customized manner according to individual characteristics.

[0007] In addition, although the prior art described in Patent Document 3 can collect microorganisms from human feces, there is no knowledge regarding the use of the collected microorganisms, and no technology for improving gut bacteria in a customized manner according to individual characteristics is disclosed at all.

[0008] The applicant of this patent application focuses on protecting or activating the beneficial bacteria possessed by an individual from among the gut microbiota for the purpose of creating a suitable intestinal environment for humans, and provides an intestinal bacteria bank business as an effort for that purpose. The intestinal bacteria bank business is one of the services that focuses on an individual's microbiome, analyzes the individual's gut microbiota, collects, proliferates, and stores the beneficial bacteria contained in the gut microbiota, and provides the beneficial bacteria or supplements using the same.

[0009] The present disclosure provides a technique that contributes to the preservation and use of gut bacteria.

MEANS FOR SOLVING THE PROBLEM

[0010] A first aspect of the present disclosure is a method for using intestinal bacteria that utilizes useful bacteria in intestinal bacteria, including a bacterial flora analysis step of analyzing the bacterial flora of feces collected from a human, an isolation step of isolating useful bacteria from a fecal collection liquid obtained by mixing the feces collected from the human into a fecal collection kit containing a predetermined bacterial preservation solution and a stirring ball and allowing a predetermined period of time to elapse, a culturing step of culturing the isolated useful bacteria, a selection step of selecting excellent useful bacteria from among the useful bacteria obtained in the culturing step, a storage step of storing the selected useful bacteria, and a useful bacteria utilization substance production step of obtaining a useful bacteria utilization substance containing at least any one of the stored useful bacteria, or a metabolite or extract of the useful bacteria, wherein the bacterial preservation solution contains a reducing agent such as L-cysteine hydrochloride, a cryoprotectant such as glycerol, and a gelling agent in a buffer solution such as Dulbecco's phosphate buffered saline, and has a pH adjusted to 3.5 to 5 with a pH adjuster such as sodium hydrogen carbonate, and is in a gel state that does not flow when left for a predetermined time.

[0011] Since the first aspect has a flora analysis step of analyzing the flora of feces collected from a human, information on what bacteria the human has in the body can be obtained by the flora analysis. Since it has an isolation step of isolating useful bacteria from a fecal collection liquid obtained by mixing the feces collected from the human into a fecal collection kit containing a predetermined bacterial preservation solution and a stirring ball after a predetermined period of time has elapsed, useful bacteria possessed by the human can be isolated and obtained. Since it has a culturing step of culturing the isolated useful bacteria, the isolated useful bacteria can be increased. Since it has a selection step of selecting excellent useful bacteria from among the useful bacteria obtained in the culturing step, the effects possessed by that type of bacteria can be exerted at a high level. Since it has a storage step of storing the selected useful bacteria, the selected useful bacteria can be used at a desired time. Since it has a useful bacteria utilization substance production step of obtaining a useful bacteria utilization substance containing at least any one of the stored useful bacteria, or a metabolite or extract of the useful bacteria, various beneficial products based on the useful bacteria can be obtained. Since the bacterial preservation solution is a gel-like substance that does not flow when left for a predetermined time, which contains a reducing agent such as L-cysteine hydrochloride, a cryoprotectant such as glycerol, and a gelling agent in a buffer solution such as Dulbecco's phosphate buffered saline and has a pH adjusted to 3.5 to 5 with a pH adjuster such as sodium hydrogen carbonate, the intestinal bacteria in the feces can be maintained until the subsequent isolation step.

[0012] The second aspect of the present disclosure is a method for using intestinal bacteria that uses useful bacteria among intestinal bacteria, including an isolation step of isolating useful bacteria from a fecal collection liquid obtained by mixing feces collected from a human into a predetermined bacterial preservation solution after a predetermined period of time has elapsed. The bacterial preservation solution is a gel-like substance that does not flow when left for a predetermined time, which contains a reducing agent such as L-cysteine hydrochloride, a cryoprotectant such as glycerol, and a gelling agent in a buffer solution such as Dulbecco's phosphate buffered saline and has a pH adjusted to 3.5 to 5 with a pH adjuster such as sodium hydrogen carbonate. This is a method for using intestinal bacteria that uses the useful bacteria.

[0013] The second aspect of the present disclosure has an isolation step of isolating useful bacteria from a fecal collection liquid in which the feces collected from the human are mixed into a predetermined bacterial preservation solution and a predetermined period of time has elapsed, so that useful bacteria possessed by the human can be isolated and obtained. The bacterial preservation solution contains a reducing agent such as L-cysteine hydrochloride, a cryoprotectant such as glycerol, and a gelling agent in a buffer solution such as Dulbecco's phosphate buffered saline, and the pH is adjusted to 3.5 to 5 with a pH adjuster such as sodium hydrogen carbonate. Since it is in a gel state that does not flow when left for a predetermined time, intestinal bacteria in feces can be maintained until the subsequent isolation step, and products using the useful bacteria can be used, such as manufacturing.

[0014] The third aspect of the present disclosure is a method for using the intestinal bacteria, wherein the bacterial preservation solution has a storage elastic modulus and a loss elastic modulus in a linear region when the strain is changed at 30°C and 1 Hz, and the magnitude of the strain (%) is 0.063 to 39.8%. The third aspect of the present disclosure is that since the bacterial preservation solution has a storage elastic modulus and a loss elastic modulus in a linear region when the strain is changed at 30°C and 1 Hz, and the magnitude of the strain (%) is 0.063 to 39.8%, it is in a gel state and easy to handle, and is also easy to mix with feces.

[0015] The fourth aspect of the present disclosure is a method for using the intestinal bacteria, wherein the bacterial preservation solution has a gelling agent content of 0.05 to 0.35% (w / v). The fourth aspect of the present disclosure is that since the bacterial preservation solution has a gelling agent content of 0.05 to 0.35% (w / v), a gel state that does not flow when left for a predetermined time is obtained, the handleability is improved, and the preservability of bacteria is also improved.

[0016] The fifth aspect of the present disclosure is a method for using the intestinal bacteria, wherein the fecal collection liquid is obtained by mixing the feces into the fecal collection kit containing the bacterial preservation solution and allowing 10 to 14 days to elapse. In the fifth aspect of the present disclosure, since the fecal collection liquid is obtained by mixing the feces into the fecal collection kit containing the bacterial preservation liquid and allowing 10 to 14 days to elapse, the desired useful bacteria can be retained in the fecal collection liquid without being killed even during a long period of 10 to 14 days.

[0017] The sixth aspect of the present disclosure is a method for using intestinal bacteria, wherein useful bacteria found to be possessed by the human in the flora analysis step are isolated in the isolation step. In the sixth aspect of the present disclosure, since useful bacteria found to be possessed by the human in the flora analysis step are isolated in the isolation step, useful bacteria present in the body of the human himself / herself can be isolated. Therefore, bacteria derived from the intestinal flora of the human himself / herself can be used, there is little risk of disturbing the intestinal flora, and the usefulness of the intestinal flora can be enhanced.

[0018] The seventh aspect of the present disclosure is a method for using intestinal bacteria, wherein in the flora analysis step, the obtained results of the intestinal flora are clearly indicated and displayed as belonging to which type among a plurality of types classified based on, for example, what kind of intestinal bacteria are abundant. In the seventh aspect of the present disclosure, since in the flora analysis step, the obtained results of the intestinal flora are clearly indicated and displayed as belonging to which type among a plurality of types classified based on, for example, what kind of intestinal bacteria are abundant, the human can easily understand what kind of intestinal flora he / she has. And based on the characteristics, guidelines on how to improve the intestinal flora can be provided.

[0019] The eighth aspect of the present disclosure is a bacterial preservation liquid in which useful bacteria can be isolated after a predetermined period has elapsed by mixing feces collected from a human. The bacterial preservation liquid contains a reducing agent such as L-cysteine hydrochloride, a cryoprotectant such as glycerol, and a gelling agent in a buffer solution such as Dulbecco's phosphate-buffered saline, and the pH is adjusted to 3.5 to 5 with a pH adjuster such as sodium hydrogen carbonate, and it is in a gel state that does not flow when left for a predetermined time. The eighth aspect of the present disclosure is a bacterial preservation solution in which useful bacteria can be isolated after a predetermined period of time by mixing feces collected from a human. The solution contains a reducing agent such as L-cysteine hydrochloride, a cryoprotectant such as glycerol, and a gelling agent in a buffer solution such as Dulbecco's phosphate-buffered saline, and the pH is adjusted to 3.5 to 5 with a pH adjuster such as sodium hydrogen carbonate. It is a gel-like bacterial preservation solution that does not flow when left for a predetermined time. Therefore, useful bacteria derived from the human gut microbiota can be obtained from the feces of that human and maintained in a viable state for a long time. Also, since it is a non-flowing gel, it is easy to handle, and it is difficult for the bacterial preservation solution to spill out of the container when collecting feces, and it is difficult to reduce the amount of the bacterial preservation solution required to maintain the bacteria in the feces. Moreover, it is also suitable for maintaining useful bacteria in feces compared to the case of a liquid with fluidity.

[0020] The ninth aspect of the present disclosure is the above-mentioned bacterial preservation solution in which the storage elastic modulus and the loss elastic modulus when the strain is changed at 30 °C and 1 Hz are in the linear region, and the magnitude of the strain (%) is 0.063 to 39.8%. The ninth aspect of the present disclosure is the above-mentioned bacterial preservation solution in which the storage elastic modulus and the loss elastic modulus when the strain is changed at 30 °C and 1 Hz are in the linear region, and the magnitude of the strain (%) is 0.063 to 39.8%. Therefore, when mixed with feces, it is easy to mix with the feces and is excellent in protecting bacteria in the feces.

[0021] The tenth aspect of the present invention is a bacterial preservation solution in which the amount of the gelling agent contained is 0.05 to 0.35% (w / v). The tenth aspect of the present invention is the above-mentioned bacterial preservation solution in which the amount of the gelling agent contained is 0.05 to 0.35% (w / v). Therefore, it becomes a non-flowing gel state when left for a predetermined time, improving the handleability, and also improving the storage stability of bacteria.

[0022] The eleventh aspect of the present disclosure is the above-mentioned bacterial preservation solution in which viable bacteria of the useful bacteria can be obtained from feces that have been mixed with feces and have passed 10 to 14 days in a fecal collection kit. According to the 11th aspect of the present disclosure, among fecal collection kits, since the bacterial preservation solution is such that viable bacteria of the useful bacteria can be obtained from feces that have been mixed and passed 10 to 14 days, it is possible to keep the bacteria in feces alive over a long period of 10 to 14 days. It is a bacterial preservation solution that is excellent not only for short-term protection within one week but also for long-term protection.

[0023] The 12th aspect of the present disclosure is a fecal collection kit that includes 30 to 90 vol% of the bacterial preservation solution in a container for containing the bacterial preservation solution. According to the 12th aspect of the present disclosure, since the fecal collection kit includes 30 to 90 vol% of the bacterial preservation solution in a container for containing the bacterial preservation solution, it is possible to reduce the amount of air taken into the fecal collection kit during fecal collection and minimize the impact on anaerobic bacteria in feces. It is a fecal collection kit that is excellent in protecting useful bacteria.

[0024] Another aspect of the present disclosure is to prepare a bacterial preservation solution in which a buffer solution such as Dulbecco's phosphate-buffered saline contains a reducing agent such as L-cysteine hydrochloride and a cryoprotectant such as glycerol, and the pH is adjusted to 3.5 to 5 with a pH adjuster such as sodium hydrogen carbonate. A method for obtaining useful bacteria is to culture a fecal collection solution in which 1 to 2 g of feces is mixed into 5 to 10 mL of the bacterial preservation solution and a predetermined period has elapsed in a medium such as M2GSC medium to isolate useful bacteria such as Lactobacillus.

[0025] According to the above-mentioned another aspect, since a bacterial preservation solution in which a buffer solution such as Dulbecco's phosphate-buffered saline contains a reducing agent such as L-cysteine hydrochloride and a cryoprotectant such as glycerol, and the pH is adjusted to 3.5 to 5 with a pH adjuster such as sodium hydrogen carbonate is prepared, and a fecal collection solution in which 1 to 2 g of feces is mixed into 5 to 10 mL of the bacterial preservation solution and a predetermined period has elapsed is cultured in a medium such as M2GSC medium to isolate useful bacteria such as Lactobacillus, useful bacteria derived from the intestinal flora of an individual can be obtained from the feces of that individual. As a result, it can be used in the future to maintain and improve the intestinal flora.

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

[0027] Since the above-mentioned another aspect is a bacterial preservation solution containing L-cysteine hydrochloride and glycerol in Dulbecco's phosphate-buffered saline, and adjusted to a pH of 3.5 to 5 with sodium bicarbonate, the collected feces can be preserved, and the useful bacteria present in the feces can be protected until they can be cultured after a predetermined period of time has elapsed.

[0028] Another aspect 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 with respect to a total of 10 mL of a buffer solution and a cryoprotectant.

[0029] Since the above-mentioned another aspect 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 with respect to a total of 10 mL of a buffer solution and a cryoprotectant, it can be kept within a predetermined pH even after being mixed with feces, and can be a bacterial preservation solution suitable for retaining useful bacteria.

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

[0031] Since the above-mentioned another aspect is a bacterial preservation solution containing a reducing agent and a cryoprotectant in a buffer solution, and adjusted to a pH of 3.5 to 5 with a pH adjuster, it can be kept within a predetermined pH even after being mixed with feces, and can be a bacterial preservation solution suitable for retaining useful bacteria.

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

[0033] Since the other aspect is a fecal collection kit comprising 5 to 10 mL of any of the above bacterial preservation solutions, even if a desired amount of feces is collected and mixed with the bacterial preservation solution, the pH of the fecal collection solution in this fecal collection kit can be adjusted to a desired pH, and the beneficial bacteria present in the feces can be protected until they can be cultured after a predetermined period of time.

[0034] Another aspect of the present disclosure is a method for isolating beneficial bacteria in which a fecal collection solution is cultured in an M2GSC medium to isolate Lactobacillus, and cultured in a paromomycin-containing TOS propionic acid agar medium to isolate Bifidobacterium.

[0035] Since the other aspect is a method for isolating beneficial bacteria in which a fecal collection solution is cultured in an M2GSC medium to isolate Lactobacillus, and cultured in a paromomycin-containing TOS propionic acid agar medium to isolate Bifidobacterium, Lactobacillus and Bifidobacterium, which are beneficial bacteria derived from the individual who produced the feces, can be isolated from the feces.

[0036] Another aspect of the present disclosure is a method for isolating beneficial bacteria in which a fecal collection solution is cultured in an M2GSC medium or the like to isolate beneficial bacteria such as Lactobacillus.

[0037] Since the other aspect is a method for isolating beneficial bacteria in which a fecal collection solution is cultured in an M2GSC medium or the like to isolate beneficial bacteria such as Lactobacillus, the necessary beneficial bacteria can be isolated from the fecal collection solution.

[0038] Another aspect of the present disclosure is a method for utilizing intestinal bacteria obtained from a subject, comprising the steps of isolating beneficial bacteria from a fecal collection solution in which feces collected from the subject are mixed in a bacterial preservation solution and a predetermined period of time has elapsed, cryopreserving the isolated beneficial bacteria, culturing the cryopreserved beneficial bacteria, and obtaining a composition containing at least one of the cultured beneficial bacteria, metabolites, or extracts of the cultured beneficial bacteria.

[0039] The other aspect is a method of using intestinal bacteria obtained from a subject, which includes a step of isolating useful bacteria from a fecal collection fluid in which feces collected from the subject are mixed in a bacterial preservation solution and a predetermined period of time has elapsed, a step of cryopreserving the isolated useful bacteria, a step of culturing the cryopreserved useful bacteria, and a step of obtaining a composition containing at least any one of the cultured useful bacteria, metabolites, or extracts of the cultured useful bacteria. Therefore, since it is a method of using intestinal bacteria, useful bacteria actually possessed by the subject in their body can be used. And it is possible to produce a composition such as a capsule, supplement, or preparation containing a substance derived from the useful bacteria.

Advantages 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 still another aspect of the present disclosure, suitable feces for obtaining useful bacteria can be secured and protected. According to still another aspect of the present disclosure, useful bacteria can be isolated. According to still another aspect of the present disclosure, uses such as manufacturing a product based on useful bacteria can be carried out.

Brief Description of the Drawings

[0041]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0042] One aspect of the present disclosure will be described with reference to the drawings based on the illustrated embodiments. The following embodiments do not unduly limit the content of the present invention described in the claims. Also, not all of the configurations described in this embodiment are essential as means for solving the present invention.

[0043] All embodiments and selectable embodiments included in the present disclosure may be combined with each other to form new embodiments. Also, all technical features and selectable technical features included in the present disclosure can form new technical features by combining them with each other.

[0044] The term "or" used in the present 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" each satisfy "A or B".

[0045] In this specification and the claims, when described as "first", "second", and "n (where n is a natural number)", it is used to distinguish different elements and is not intended to indicate a specific order or superiority or inferiority. For configurations common to each embodiment, the same reference numerals are given and duplicate descriptions are omitted.

[0046] The terms such as "above", "below", "front", "rear", "left", "right", etc. used in this specification and the claims, and the directions or positional relationships represented by terms including these terms are based on the drawings and are only for conveniently and briefly explaining the embodiments. Therefore, unless there are clear definitions or limitations, it should be understood that the specific elements and their use do not explicitly or implicitly indicate that they are configured in a specific direction, nor do they limit the claims and embodiments.

[0047] For numerical values or elements modified by terms such as "about", "approximately", "substantially", and "essentially" used in this specification and the claims, they are understood to include the numerical value itself and the numerical values before and after centered around that numerical value, and also to include the element and what can be said to be the same as that element. For example, when described as "about 3", if the technical features and technical significance claimed in this disclosure are not different, "3" and the numerical values consecutive to it may also be included in "about 3". Also, in the case of "B that is substantially the same as A", in addition to the case where B is completely identical to A, as long as the technical features and technical significance claimed in this disclosure are common, even if there are differences, they may be included within the scope of "substantially".

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

[0049] Hereinafter, one aspect of the present disclosure will be specifically described.

[0050] 1. Microbiota analysis In the human body, there are bacteria that always inhabit the human body, called human resident bacteria. These bacteria are diverse and vary depending on each organ and tissue of the human body. For example, on the skin, there are skin resident bacteria that adapt to the environment exposed to the outside world, and in digestive organs such as the intestine, anaerobic bacteria exist because oxygen is difficult to reach. Conventional approaches to these human resident bacteria have aimed to study bacteria that cause diseases and health deterioration in the human body, such as pathogenic bacteria, and eliminate or suppress these bacteria. However, elucidating only pathogenic bacteria that cause harm to the human body could not truly contribute to human health. For example, in today's era of reaching 100 years of life, especially since the goal is not just to live long but to live a healthy long life, it has become necessary to be healthy on a daily basis without getting sick, and studying and taking measures against pathogenic bacteria alone is insufficient.

[0051] In recent years, research in the field of genetics has advanced, and its analysis techniques have improved. As an example, the emergence of the Next Generation Sequencer has made it possible to analyze the bacterial flora, which was previously difficult. Using the technology of this next-generation sequencer, bacteria that have been difficult to culture can be analyzed. From another perspective, it has become possible to analyze the bacterial flora, including not only bacteria that function poorly in the human body, such as pathogenic bacteria, but also bacteria that contribute to health promotion and bacteria that contribute to both (opportunistic bacteria). When the bacterial flora containing such various bacteria becomes visible, it has been found to be very useful for understanding the health status of humans. Therefore, focusing on the fact that the state of the intestinal flora is closely related to the maintenance and promotion of human health, and that such analysis of the intestinal flora can be performed by using the next-generation sequencer, the following-described service was developed with the intention of making the above findings more useful on a personal basis.

[0052] 2. Intestinal Flora Analysis Service

[0053] Since the intestinal flora of each person is different and not the same for everyone, it is first considered necessary to analyze what the intestinal flora of a specific individual is like. Therefore, it is preferable to collect the feces of an individual and analyze the bacterial flora contained in the feces. The reason for analyzing human feces for the analysis of the intestinal flora is that intestinal bacteria, both live and dead, are excreted outside the body as feces, so they can be easily collected without using surgical techniques on the human body.

[0054] For the analysis of the gut microbiota, it is preferable to use the next-generation sequencer described above. This is because the next-generation sequencer can simultaneously determine the gene sequences in DNA or RNA samples of multiple types of bacteria, and bacterial classification and identification can be performed based on the nucleotide sequences of bacterial genes, their descriptions, and the nucleotide sequence databases in which literature information is stored. Since the conventional Sanger method can only analyze one gene sequence of one type of bacteria, the use of the next-generation sequencer is excellent for analyzing the 16S rRNA gene of the gut microbiota, which contains a vast amount of genetic information.

[0055] In the analysis by the next-generation sequencer, it has become mainstream to classify the gut bacteria in feces at the levels of "phylum", "genus", and "species". When classifying at the "phylum" level, since "genus" and "species" are grouped into one category, it becomes difficult to distinguish the characteristics of individual bacterial species and strains based only on the "phylum" notation. Also, although it is possible to distinguish the characteristics of individual bacteria by classification at the "species" level, the analysis using the 16S rRNA gene by the next-generation sequencer with a low classification rate lacks accuracy in classification. Classification at the "genus" level can distinguish the characteristics of individual bacteria compared to "phylum", and it is possible to classify with a higher classification rate than "species". From the above, the "genus" level is preferable when classifying the gut microbiota.

[0056] The analysis results are obtained in terms of the names of the classified bacteria and their numbers or occupancy rates, but simply listing them randomly as a table does not increase their utility value. Therefore, these results can be presented as multiple outputs from various perspectives. As one output format, it shows the names of bacteria classified into several, for example, 10 genera in descending order of the number of bacteria contained in feces, along with the characteristics and functions of those bacteria. This is because it is considered that the influence of the top bacteria is strongly reflected in the human body.

[0057] As another output mode, it shows a list of bacteria regarded as useful bacteria and a list of bacteria regarded as harmful bacteria among the bacteria contained in feces, along with their characteristics and functionality. This is because it enables one to know what kinds of useful and harmful bacteria an individual has, that is, to know their names. As yet another output mode, it assigns the types and quantities of various bacteria to several pre-defined evaluation categories, and shows the categories and what they represent.

[0058] Although the types and abundance ratios of the bacteria that make up the gut microbiota vary from person to person, it is beneficial as an initial evaluation of the gut microbiota to be able to classify them into several types according to certain characteristics and point out the characteristics of each type. One example is the classification method that divides them into five types: BA, BF, F, R, and P.

[0059] These five types are classified according to the types of bacteria with high occupancy rates in the gut. The BA type is a type in which many Bacteroides bacteria, which are known to have a beneficial effect on lipid metabolism, are present. The BF type is a type in which many Bifidobacterium bacteria, which produce acetic acid and lactic acid, maintain a low intestinal pH, and suppress bacteria such as Escherichia coli to regulate the intestinal environment, are present. The F type is a type in which many Faecalibacterium bacteria, which are said to produce butyric acid and contribute to disease prevention and improved defecation, are present. The R type is a type in which many Lachnococcus bacteria, which are said to have the function of decomposing water-soluble dietary fiber to produce short-chain fatty acids related to immunity, are present. The P type is a type in which many Prevotella bacteria, which decompose dietary fiber and produce succinic acid and acetic acid, are present.

[0060] And yet another output mode is to present a list of the names of bacteria classified by "genus" or "species" present in feces in descending order of quantity. By listing all the analyzed bacteria, it is possible to display without omission the bacteria with unknown characteristics and the minority bacteria. Alternatively, based on the accumulated analysis data such as flora and short-chain fatty acids, it is also possible to display the abundance ratios of some beneficial bacteria, harmful bacteria, and short-chain fatty acids in feces. Such data can be used to improve the individual's intestinal environment and diet.

[0061] In this way, through the flora analysis of the intestinal flora from an individual's feces, data on what kinds of bacteria and in what amounts are contained in the intestinal flora of that individual can be obtained. This data will serve as the material for the utilization of intestinal bacteria described below. That is, among the bacteria found in the individual's intestine through flora analysis, in the next step, the desired beneficial bacteria are isolated and preserved.

[0062] 3. Explanation of the method of using intestinal bacteria

[0063] The general outline of the method of using intestinal bacteria (intestinal bacteria bank) for collecting and storing useful microorganisms from an individual's intestinal flora will be explained based on the flowchart shown in Figure 1.

[0064] (1) Feces collection This is the step of collecting the bacteria that make up the human intestinal flora (ST1 in Figure 1). For this purpose, human feces are collected. The collected feces are placed in a predetermined feces collection kit containing a predetermined bacterial preservation solution and stored, and then delivered to the bacterial isolation and culture institution.

[0065] (2) Flora analysis (flora analysis step) This is the step of analyzing the types and composition ratios of the bacteria collected from feces (ST2 in Figure 1). By performing flora analysis on the specimen obtained from feces, it is possible to analyze whether a predetermined beneficial bacterium is contained in the bacteria of that individual and its quantity.

[0066] Note that, as described in 1 above, this flora analysis can be performed separately in advance, and the flora analysis performed at this stage can also be omitted.

[0067] (3) Isolation of beneficial bacteria This is a step of isolating desired beneficial bacteria from a fecal collection fluid that has been in a fecal collection kit for a predetermined period (ST3 in FIG. 1). Since various bacterial species such as beneficial bacteria and harmful bacteria exist in feces, beneficial bacteria are searched for and identified among them.

[0068] (4) Culturing of isolated bacteria (culturing step) This is a step of culturing the isolated beneficial bacteria (ST4 in FIG. 1). The identified beneficial bacteria are cultured to increase the number to an appropriate level.

[0069] (5) Selection of stored bacteria This is a step of selecting suitable strains among the cultured bacteria of the same species (ST5 in FIG. 1). This is to collect better strains considering factors such as ease of growth and resistance.

[0070] (6) Storage of selected bacteria (storage step) This is a step of storing the desired selected bacteria (ST6 in FIG. 1). The selected bacteria are mixed with a storage solution, placed in a predetermined tube, and cryopreserved so that they can be taken out and used when needed.

[0071] (7) Use of stored bacteria (manufacturing process of beneficial bacteria utilization substances) This is a step of using the stored beneficial bacteria (ST7 in FIG. 1). The frozen bacteria are thawed, cultured, and increased, and used for various purposes. Various beneficial bacteria utilization substances are manufactured, including the beneficial bacteria themselves, extracts from the beneficial bacteria, capsules, tablets, and other supplements containing metabolites of the beneficial bacteria. And the beneficial bacteria utilization substances are provided to the individual who is the source of the beneficial bacteria or others who want to increase such beneficial bacteria.

[0072] (8) Provision of information on intestinal flora This is a step of providing information on intestinal bacteria (ST8 in FIG. 1). The characteristics of the intestinal flora of an individual are found from the analysis based on flora analysis and the information is provided and utilized. For example, by analyzing the initial flora analysis and the changes in the intestinal flora after changes in diet or intake of supplements, the effects of diet changes and supplements can be verified.

[0073] The method of using intestinal bacteria (intestinal bacteria bank) according to one aspect of the present disclosure is performed as described above. However, the method of using the intestinal bacteria 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 the steps may be interchanged if there is no technical inconsistency.

[0074] In addition, the method of using intestinal bacteria (intestinal bacteria bank) can be configured such that an individual performs it only once, or can also be configured to perform it multiple times over time. When an individual continuously implements the method of using intestinal bacteria, one or more intestinal bacteria (intestinal flora, including the collected feces) collected according to the individual's situation at that time can be stored, or the stored ones can be used over time. In that case, by comparing the analysis information of the flora analysis performed at intervals, it can be used for improving health status and preventing diseases.

[0075] 4. Description of technologies that can constitute the method of using intestinal bacteria

[0076] Examples of embodiments of various technologies that can be used to implement the above method of using intestinal bacteria (intestinal bacteria bank) will be described.

[0077] <Feces collection step> Since each individual has a different intestinal flora, it is necessary to collect the bacteria for each individual. And in order to collect the bacteria of that individual (human) without difficulty without going through a surgical process or the like, it is preferable to collect them from the feces of that individual.

[0078] (1) Feces collection kit For collecting feces, it is preferable to use a feces collection kit suitable for the intestinal bacteria bank. The first reason is that there is a time blank from collecting feces to collecting the bacteria in the feces, and it is necessary to prevent the bacteria from dying during that time. The second reason is that it is necessary to be suitable for storage and transportation from feces collection. The feces collection kit has a bacteria preservation solution sealed in a predetermined container.

[0079] Bacterial preservation solution: First, the bacterial preservation solution enclosed in the feces collection kit needs to preserve the bacterial flora contained in the feces and keep the bacteria from dying for a predetermined period until the next step. The "predetermined period" is the period from when the feces are put into the feces collection kit until the isolation of useful bacteria is started at the testing institution. 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 1 week or around 1 week. The reasons include the number of days required for transporting the collected feces to the location of the testing institution, and the fact that even when the feces collection kit containing the feces reaches the testing institution, it may take some days until the collection and culturing of microorganisms are carried out. From these perspectives, the following bacterial preservation solution was developed.

[0080] This bacterial preservation solution is a liquid containing a reducing agent and a cryoprotectant in a buffer solution and adjusted to a pH of 3.5 to 5 with a pH adjuster. Since bifidobacteria and lactic acid bacteria produce organic acids, they have stronger acid resistance compared to other bacteria. However, the pH suitable for their growth is not acidic, and the optimum pH is neutral at 7 to 8. However, the pH is adjusted to weakly acidic to exert the effect of reducing agents such as L-cysteine hydrochloride described later.

[0081] The buffer solution has the function of suppressing the influence of pH. Specific buffer solutions 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, etc. More specifically, the buffer solution is preferably phosphate buffered saline (PBS) or D-PBS containing potassium chloride (Dulbecco's Phosphate Buffered Saline) and more preferably Dulbecco's phosphate buffered saline without divalent cations (D-PBS(-) ) is is even more preferred. D-PBS(-) isThe more preferable reason is that it can minimize the pH fluctuation of the bacterial preservation solution due to the mixing of feces and keep the bacteria in the feces alive without damage. Note that the "Dulbecco's phosphate buffered saline or the like" described in the claims may include the various buffer solutions exemplified above.

[0082] The reducing agent is a component for not inhibiting the growth of anaerobic bacteria such as Bifidobacterium and lactic acid bacteria, and also contributes to the growth improvement of lactic acid bacteria. Examples of the reducing agent include L-cysteine, N-acetylcysteine, methylene blue, etc. L-cysteine hydrochloride is used not only as a food additive but also for reducing oxygen in water to create anaerobic conditions.

[0083] The pH adjuster is used to relieve the stress on the bacteria in the feces by adjusting a 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 - 5). Examples of the pH adjuster include carbonates, bicarbonates, hydrochloric acid, sodium hydroxide, etc., but are not limited thereto. More specifically, sodium bicarbonate and sodium carbonate are preferable as the pH adjuster, and among them, sodium bicarbonate is more preferable. The reason is that sodium bicarbonate is also suitable for the growth of anaerobic bacteria, methane bacteria, and is easy to finely adjust the pH. In contrast, hydrochloric acid and sodium hydroxide are classified as highly toxic substances, while sodium bicarbonate is generally used as baking soda and thus has high safety. Note that the "sodium bicarbonate or the like" described in the claims may include the various pH adjusters exemplified above.

[0084] The cryoprotectant can impart suitable viscosity to the preservation solution. Specifically, glycerol and dimethyl sulfoxide (DMSO) can be exemplified, but are not limited thereto. Glycerol is also preferable in that it functions as an emulsifier, a stabilizer, and an anti-curing agent. In addition, glycerol is preferred because it is less expensive than the organic solvent dimethyl sulfoxide and is not dangerous to the human body, and thus is widely used in bacterial preservation. Note that "glycerol etc." described in the claims may include the various preservation solutions exemplified above.

[0085] It is preferable to prepare the bacterial preservation solution so that the pH of the bacterial preservation solution is 3.5 or more and 5 or less. When the pH of the fecal collection solution after putting the required amount of feces into the fecal collection kit and mixing it with the bacterial preservation solution is 5 or more, useful bacteria survive. Therefore, in order to mix the feces and make the pH 5 or more, the pH of the bacterial preservation solution may be 4-5 or 3.5-5.

[0086] In other words, if the pH adjuster is not added to adjust the pH, the bacterial preservation solution will become strongly acidic (pH 2 or more and less than 3), and this state is quite stressful for intestinal bacteria. However, in order to reduce the stress on intestinal bacteria while maintaining the effect of the reducing agent, it is adjusted to weak acidity (pH 3 or more and less than 6, preferably pH 3.5-5).

[0087] The content of each component in the bacterial preservation solution is 0.005 or more and 0.02 or less of the reducing agent with respect to a total of 10 mL of the buffer solution and the cryoprotectant. If it is less than 0.005 g, there is no reducing effect, and if it is more than 0.02 g, the pH becomes too low.

[0088] The content of the pH adjuster can be 0.5 mg or more and 30 mg or less, can be 0.5 mg or more and 10 mg or less, and can be 0.5 mg or more and 2 mg or less with respect to a total of 10 mL of the buffer solution and the cryoprotectant. If it is less than 0.5 mg, it is difficult to adjust the pH, and if it is less than 5 mg, the pH may not be adjustable. If it exceeds 2 mg, 10 mg, or 30 mg, the effects of other components may be weakened.

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

[0090] As a preferred embodiment of the bacterial preservation solution, there can be mentioned a bacterial preservation solution obtained by mixing 5 mL of a 50% glycerin solution, 5 mL of D-PBS(-), 0.01 g of L-cysteine hydrochloride, and 0.5 mg or more and 10 mg or less, or 0.5 mg or more and 2 mg or less of sodium hydrogen carbonate.

[0091] When feces are put into a general feces preservation solution for preservation, anaerobic useful bacteria often die. However, according to the above bacterial preservation solution, it is suitable for the preservation of anaerobic bacteria, and after a user, who is an individual, collects feces, the possibility of death of useful bacteria can be reduced until it is received by a service provider such as an operator of this intestinal flora bank.

[0092] In another aspect of the bacterial preservation solution, it further contains a gelling agent in the above bacterial preservation solution. And this bacterial preservation solution has the property that when left standing for a predetermined time, it becomes a gel state that does not flow, and when mixed with feces and shaken, it becomes a liquid state with fluidity. "When left standing for a predetermined time, it becomes a gel state that does not flow" means that when left at room temperature for at least one day, it becomes a gel state that has lost fluidity and changes to a sol state with fluidity by shaking. Also, when mixed with feces and shaken, the bacterial preservation solution and feces are mixed to form a feces collection solution, and this feces collection solution is in a sol state with fluidity. The feces collection solution is in the state of the mixed feces, and the property after storage after shaking may be in a gel state 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 elodeaIt is a purified product obtained by deacetylating the polysaccharide produced by [[ID=]], and mainly composed of glucose, rhamnose, and uronic acid, and may be suitable for culturing microorganisms that are difficult to culture. In contrast, when, for example, agar is used as a gelling agent, it may interfere with culturing, such as suppressing the swarming of certain microorganisms. The reason why gellan gum is suitable for culturing microorganisms that are difficult to culture is that, in addition to the above-mentioned reason that the bacterial preservation solution has an appropriate viscosity and gently acts on the environmental changes of bacteria, it may act on extracellular enzymes related to the uptake of nutrients, etc., and may have an auxiliary effect on difficult-to-culture bacteria by acting outside the bacterial cells.

[0096] Container: The fecal collection kit is composed of a container and a bacterial preservation solution contained therein.

[0097] The container is preferably composed of a container body and a lid. The container body is bottomed and has an opening at the top, and is made of glass or plastic that can enclose the bacterial preservation solution. The lid seals the opening of the container body, and it is preferably provided with a spoon-shaped spatula inside the lid for scraping feces and putting them into the container body. The spatula preferably has a recess that can hold about 1 to 2 g of feces. This is because 1 to 2 g of feces can be collected by scraping feces at the tip of the spatula and placing the feces on the tip. However, since a fairly large recess is required to hold 1 to 2 g of feces, the size of the recess can be set to be able to hold about half or one-third of 1 to 2 g of feces, and feces can be collected two or three times.

[0098] Alternatively, it may be provided with an outer container for further enclosing the container composed of the container body and the lid. By providing the outer container, leakage of the fecal collection liquid to the outside can be strengthened.

[0099] An example of a feces collection kit is shown in the schematic perspective view of FIG. 3. This feces 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 into which a bacterial preservation solution 40 is placed, and a lid 22 for sealing the bacterial preservation solution 40 in the inner container body 21. The spoon-shaped spatula 23 attached to the lid 22 has a recess 25 at the tip of its handle 24 for placing feces, and a baffle plate 26 for preferably mixing feces into the bacterial preservation solution 40 in the vicinity of the recess 25 of the handle 24. The recess 25 portion may be bent from the handle 24 portion or may extend straight from the handle 24 portion.

[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 configured to be placed in the outer container 30, even if the feces collection liquid in which feces are mixed with the bacterial preservation solution 40 leaks outside the inner container 20 by any chance, leakage to the outside from the outer container 30 can be prevented, which is hygienic, and leakage of the smell of feces can also be prevented. Commercially available containers such as those manufactured by Zalstat can be used for such inner containers 20 and outer containers 30.

[0101] It is preferable to put a predetermined amount of stirring balls 27 in the inner container 20. Since the bacterial preservation solution 40 becomes gel-like and has no fluidity after being left for a while, it is preferable to shake and mix the inner container 20 after putting feces. However, if the stirring balls 27 are in the inner container 20, it helps to fluidize the bacterial preservation solution and facilitates the mixing of the bacterial preservation solution and feces. The stirring balls 27 contribute to mixing the bacterial preservation solution and the feces, and may be any suitable ones for promoting the 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 put 3 to 10 of them into the inner container 20. The stainless steel balls do not change the properties of the fecal collection solution, and by putting 3 to 10 balls with a diameter of 2 to 4 mm, the stirring efficiency can be made excellent. If it is smaller than 2 mm or larger than 4 mm, or less than 3, the stirring efficiency will deteriorate. Also, if the size is larger than 4 mm or more than 10 are put in, the liquid volume of the bacterial preservation solution 40 will be relatively small, and it will also lead to an increase in cost, which is not preferable. Making the number of the stirring balls 27 be 4, 5, 6, 7, 8, or 9 and their diameter be 3 mm is one aspect of using the stirring balls 27.

[0102] The amount of feces collected is 0.5 to 3 g, preferably 1 to 2 g. If it is less than 0.5 g, there is a risk that the necessary useful bacteria cannot be collected. Also, since the necessary useful bacteria can be collected with a collection amount up to 3 g, even if feces are collected exceeding 3 g, it is necessary to increase the required amount of the bacterial preservation solution or enlarge the inner container, resulting in high cost. The liquid volume of the bacterial preservation solution included in the fecal collection kit is 5 to 20 mL, preferably 5 to 15 mL, more preferably 8 to 12 mL. This is because it is necessary to include the amount required to mix with the collected feces and obtain the bacteria in it without killing them. If it is less than 5 mL, it is too little for the amount of feces collected, and there is a risk that the useful bacteria in the feces will die. If it exceeds 20 mL, the inner container will also become larger, resulting in high cost.

[0103] It is preferable that the inner container contains a bacterial preservation solution in an amount of 30 to 90 vol% of its internal volume. If the amount is less than 30 vol%, the protection of bacteria in feces may be insufficient, and there may be many voids that are mixed in when the feces are mixed, increasing the risk of impairing the survival of anaerobic bacteria. Also, if it exceeds 90 vol%, there is a risk that the fecal collection solution will overflow from the inner container when the feces are put into the inner container. Therefore, it is preferable to select and adopt an inner container with an internal volume of 5.5 to 23 mL, preferably 12 to 18 mL, so that the feces of the above-mentioned collection amount can be put into the inner container containing the bacterial preservation solution of the above ratio and the inner container can be covered.

[0104] After collecting feces, it is preferable to shake the inner container 20 containing feces 5 to 20 times, preferably 8 to 12 times, to mix the feces sufficiently with the bacterial preservation solution. This is because if the feces are not sufficiently wrapped in the bacterial preservation solution and are exposed to air, useful bacteria may die. Note that the outer container 30 may not be necessary.

[0105] Then, after packing the fecal collection kit containing feces, it is delivered to a place where there is a predetermined institution for isolating bacteria from an individual. It is preferable to store the fecal collection kit in a refrigerator from after feces collection until delivery. Also, since it is desired to collect bacteria from feces at an early stage, it is preferable to send it by refrigeration within 2 days after feces collection.

[0106] <Step of isolating useful bacteria> This is a step of isolating a predetermined useful bacterium from among various bacteria contained in feces.

[0107] Useful bacteria refer to bacteria that have a beneficial effect on human health, such as so-called good bacteria. Examples of useful bacteria include Bifidobacterium or lactic acid bacteria. Among lactic acid bacteria, Lactobacillus ( Lactobacillus ) may be present in a small number in feces depending on the individual. Therefore, for individuals who have been confirmed to have Lactobacillus by analyzing the bacterial flora with a next-generation sequencer (NGS), the isolation of Lactobacillus is effective.

[0108] Other useful bacteria include butyric acid-producing bacteria such as Faecalibacterium ([ Faecalibacterium Faecalibacterium ), Roseburia ([ Roseburia Roseburia ), Akkermansia ([ Akkermansia Akkermansia ), Collinsella ([ Collinsella Collinsella ), bacteria other than lactic acid bacteria such as Pediococcus and Blautia ([ Blautia Blautia ).

[0109] To isolate useful bacteria, it is preferable to use a predetermined liquid medium according to the bacteria to be isolated from the fecal collection fluid. This is because isolating the desired bacteria becomes easier if the growth of bacteria other than the desired bacteria can be excluded. Such media include a medium obtained by adding a selective agent such as an antibiotic that inhibits the growth of other bacteria to a medium in which other bacteria other than the isolation target can also grow, a medium in which colonies of only the desired bacteria can be obtained by using a carbon source that can be utilized only by the desired bacteria, and a medium that utilizes properties peculiar to the desired bacteria.

[0110] When isolating Bifidobacterium as a predetermined liquid medium according to the bacteria, examples include MGLP agar medium, modified BCP-added plate count agar medium, TOS mupirocin medium, TOS propionic acid agar medium, paromomycin-containing TOS propionic acid agar medium, and the like. The MGLP agar medium and the modified BCP-added plate count agar medium can suppress the growth of lactic acid bacteria that are likely to be mixed by restricting the nutrient source. In the TOS mupirocin medium, Bifidobacterium grows well due to galactooligosaccharides in the TOS medium, while mupirocin inhibits the growth of lactic acid bacteria, so it is possible to suppress the contamination of lactic acid bacteria that are likely to be mixed with Bifidobacterium.

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

[0112] When isolating Lactobacillus, MRS medium or MRS medium containing bile acid (cholic acid) can be used. MRS medium is equipped with nutrients required by fastidious lactic acid bacteria, and ammonium citrate and sodium acetate contained therein inhibit the growth of many bacteria other than lactic acid bacteria. If MRS medium containing bile acid is used, lactic acid bacteria with high resistance to bile acid can be obtained, and lactic acid bacteria that are easily reachable to the intestine can be selectively obtained.

[0113] Alternatively, instead of using a selective medium that does not allow the growth of bacteria other than predetermined useful bacteria, it is also possible to culture using a medium in which various bacteria can grow, extract DNA from colonies having the characteristics of the desired useful bacteria from the entire obtained colonies, identify the bacterial species, and isolate the desired useful bacteria. By using a medium in which multiple bacterial species can grow, the labor of culturing in multiple media can be saved.

[0114] Examples of such media include M2GSC medium, BCP-added plate count agar medium (BCP plate medium), calcium carbonate-containing MRS agar medium, and the like. M2GSC medium is used for culturing bacteria that are difficult to culture or highly sensitive to oxygen, such as Faecalibacterium. However, since M2GSC medium contains high nutrient sources for Lactobacillus, such as glucose, fructose, and cellobiose, Lactobacillus can also grow. The BCP-added plate count agar medium makes it easy to detect lactic acid bacteria due to the yellowing of BCP (bromocresol purple) contained in the medium by lactic acid production by lactic acid bacteria, even if various bacteria are contained. The calcium carbonate-containing MRS agar medium is turbid due to the presence of calcium carbonate, but since lactic acid bacteria dissolve the calcium carbonate, the area around the colonies of lactic acid bacteria becomes transparent, making it easier to detect lactic acid bacteria.

[0115] And after culturing on these media, the desired useful bacteria can be isolated by extracting DNA from the obtained colonies and identifying the bacterial species.

[0116] <Isolation step of selected bacteria> Since bacteria belonging to the same genus also have different characteristics at the strain level, it is important to select useful bacteria to be stored even among bacteria belonging to the same Bifidobacterium or Lactobacillus in terms of obtaining highly useful bacteria. For example, if a bile acid tolerance test is performed on a plurality of isolated strains and a strain with strong resistance to bile acid is found, it is considered that the bacterium also has high resistance to gastric acid and is likely to reach the intestine. Thus, various selection steps such as a bile acid tolerance test can be added to obtain bacteria with more favorable properties.

[0117] <Preservation (storage) step of selected bacteria (accumulation step of isolated bacteria)> The isolated and identified bacterial cells can be stored by mixing them with a preservation solution together with the culture solution, putting them into a predetermined tube, and storing them frozen. The tube for storage 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 it is preferably 1.5 to 2.0 mL in capacity.

[0118] For the culture solution containing useful bacteria before mixing with the cryoprotectant, various media such as MRS medium can be used, and it is preferable that the content of the useful bacteria isolated in the medium is 1.00E+08 / mL or more, or the content until the growth curve of the useful bacteria reaches the stationary phase is included. This is because it is considered that cells in the stationary phase are more resistant to freezing stress than cells in the logarithmic growth phase. Also, since a certain proportion of cells will die due to freezing, if it is less than 1.00E+08 / mL, there may be a risk that it is difficult to grow to almost the required amount during use, and the storage efficiency of the useful bacteria is not good. And it is substantially difficult to contain more than 1.00E+08 / mL.

[0119] The content of the cryoprotectant in the mixed solution obtained by mixing such a culture solution containing useful bacteria and the cryoprotectant is 15 - 20 vol%. If the proportion of the cryoprotectant is less than 15 vol%, the culture solution may freeze during freezing and there is a risk of destroying the useful bacteria. 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 will decrease, making it difficult to grow when using this useful bacteria, and the storage efficiency is not good.

[0120] After mixing the medium containing useful bacteria and the cryoprotectant, as conditions for storing the obtained storage solution in a tube, there are storage methods at -196°C to -160°C using liquid nitrogen, or in an ultra-low temperature freezer with specifications of -150°C, -90°C to -80°C, -60°C to -40°C, -35°C to -20°C. The lower the temperature, the more beneficial it is to maintain the cessation of bacterial metabolic activity and the resting state of cells for a long time. However, if it is -196°C to -150°C, costs associated with maintaining cooling and securing a wide area are also required. Also, if it is -60°C to -20°C, it is easily affected by the ambient temperature, and when the storage is opened and closed, the temperature inside the storage fluctuates greatly, leading to deterioration of the quality of the cryopreserved sample and making it difficult to store the sample for a long time. Therefore, it is preferable to store it frozen at around -80°C using an ultra-low temperature freezer with specifications of -90°C to -80°C.

[0121] <Use of the intestinal bacteria bank> The preserved useful bacteria are cultured, and useful bacteria-related products are manufactured by encapsulating the useful bacteria themselves, extracts from the useful bacteria, metabolites of the useful bacteria, etc. in capsules or using them as raw materials for supplements. Then, for example, by returning them to the collected individual, it is possible to contribute to the improvement of the intestinal flora of that individual.

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

Example

[0123] <Experiment 1: Functional evaluation of bacterial preservation solution (Part 1)> Test objective: To evaluate the preservability of Bifidobacterium by fecal collection fluid so that the beneficial bacteria in the collected feces do not die after a predetermined period in the fecal collection fluid.

[0124] Preparation of bacterial preservation solution: D-PBS(-), glycerin solution, L-cysteine hydrochloride, and sodium hydrogen carbonate solution were mixed to prepare six types of bacterial preservation solutions 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 pH values of 3.0, 3.5, 4.0, 4.5, 5.0, and 5.5, which were designated as Samples 2 to 7, respectively. In addition, a bacterial preservation solution with unadjusted pH (NC) without adding sodium hydrogen carbonate was also prepared and designated as Sample 1. The pH of this unadjusted bacterial preservation solution was strongly acidic at 2.7 - 2.8.

[0125] Test method: Approximately the same amount of feces defecated by the same person at the same time was added to 10 mL of each of the above samples and completely suspended until the fecal lumps disappeared. Then, the fecal collection fluid on the day of feces collection and after storage at 4°C for 7 days was 10 -3 、10 -4 、10 -5Dilute stepwise by a factor of two, apply the final dilution to TOS medium, which is a dedicated agar medium for Bifidobacterium, and culture at 37 °C under anaerobic conditions for 72 hours. After culturing, count the number of colonies formed on the agar plate, and calculate the survival rate by applying the number of colonies on the day of stool collection and after 7-day storage to the following formula. Survival rate = (number of colonies after 7-day storage / number of colonies on the day of collection) In this test, samples with different stool collection days (defecation days) were prepared in the same manner, and the number of samples for each sample was set to 4.

[0126] Test results: The survival rates of the above samples were shown in the box-and-whisker plot of Figure 2. As shown in Figure 2, in Samples 3 to 6 in the pH range of 3.5 to 5.0, the median was close to the reference value of "1.0" for the survival rate, and the interval between the maximum value and the minimum value was narrow. In addition, compared with Sample 1 without pH adjustment and Sample 7 with pH 5.5, the results of the four survival rates of these samples were concentrated in the center.

[0127] On the other hand, in Sample 1 without pH adjustment, the survival rate could be 0, and the median was the farthest from the reference value of "1.0". In Sample 7 with pH 5.5, the survival rate was the highest compared to other samples, but it was considered that the survival rate increased due to the growth of bacteria other than Bifidobacterium.

[0128] From these results, it was confirmed that by using a bacterial preservation solution with the pH adjusted to "3.5 to 5", Bifidobacterium can be stored in the fecal collection solution for a predetermined period without being killed.

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

[0130] Test method An attempt was made to isolate Lactobacillus using M2GSC medium and MRS medium containing bile acid (cholic acid). A fecal collection fluid mixed with the same feces as used in Experiment 1 (Sample 5a with a pH of 4.5 after preparation of the bacterial preservation solution and a pH of 6.39 of the fecal collection fluid after mixing with feces) was added at 5.0% (v / v) to each of M2GSC medium, MRS medium, and MRS medium containing 0.05 - 1.0% (w / v) bile acid, and cultured at 37°C under anaerobic conditions for 72 hours.

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

[0132] Test results: Round yellow or round white colonies were confirmed in all three media. However, in the normal MRS medium, the number of round yellow colonies was less than that in M2GSC medium and MRS medium containing bile acid.

[0133] All the bacterial cells obtained from the six colonies selected from M2GSC medium and normal MRS medium showed high homology (98% ≤) with Lactobacillus species. However, the obtained bacterial species were biased by the medium, and Lactobacillus casei paracasei ( Lacticaseibacillus paracasei ) or Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ) preferentially grew in M2GSC medium, and Limosilactobacillus fermentum ( Limosilactobacillus fermentum ) preferentially grew in the normal MRS medium.

[0134] Needle-shaped crystals were confirmed after culturing in MRS medium containing bile acids. These crystals are metabolites produced by the decomposition of bile acids by bile salt hydrolase (BSH) possessed by lactic acid bacteria such as Lactobacillus. Since BSH-producing lactic acid bacteria are known to promote a decrease in blood cholesterol, it was also found that such lactic acid bacteria were growing in MRS medium containing bile acids. In addition, from the MRS medium containing bile acids, Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ), Lactiplantibacillus pentosus ( Lactiplantibacillus pentosus ), Lentilactobacillus senior ( Lentilactobacillus senioris ), Lactiplantibacillus fabifermentans ( Lactiplantibacillus fabifermentans ) and other lactic acid bacteria were also confirmed, and it was also found that Lactiplantibacillus plantarum was particularly abundant.

[0135] <Experiment 3: Isolation and culture of Bifidobacterium> Test purpose To examine a method suitable for isolating Bifidobacterium.

[0136] Test method Paromomycin-containing TOS propionate agar medium, which is favorable 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 filter-sterilized with a 0.22 μm syringe filter. Next, the TOS agar medium was autoclaved (115 °C, 15 minutes), then cooled to 50 °C, and the paromomycin sulfate solution was added to the TOS agar medium so that the final concentration was 50 μg / mL and mixed.

[0138] Next, the same fecal extract used in Experiment 2 was diluted 10-fold -2 、10-fold -3 、10-fold -4 、10-fold -5 、10-fold -6Dilute stepwise by a factor of two, and smear 80 μL of the final dilution on the paromomycin-containing TOS propionic acid agar medium. Then, culture this medium at 37°C under anaerobic conditions for 48 to 72 hours. Next, extract DNA from the formed single colonies, amplify the 16S rRNA gene (1.5 kb) by PCR, analyze the nucleotide sequence of the amplified PCR product using a sequencer, and perform identification and analysis of the bacterial species using the Blast search system of NCBI.

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

[0140] <Experiment 4: Preservation (Storage) of Selected Bacteria> Test objective To prepare storage tubes for storing the desired useful bacteria collected from feces, isolated, cultured, and selected.

[0141] Prepare 1.5 - 2.0 mL tubes for cryopreservation of the useful bacteria. First, for the colonies of the useful bacteria selected from those isolated from feces and purely cultured, scrape the useful bacteria with a platinum loop, inoculate them into MRS medium, and further culture them at 37°C for 48 to 72 hours under anaerobic or aerobic conditions. The anaerobic or aerobic conditions were determined according to the type of useful bacteria. Add 200 μL of a sterilized 50% glycerol solution to the tube, and then add 800 μL of the culture solution of the useful bacteria and mix. Then, attach an IC chip to this tube and store the obtained useful bacteria by cryopreservation at a condition of ≤ -80°C.

[0142] <Experiment 5: Functional Evaluation of Bacterial Preservation Solution (Part 2)> Test objective As described in the section on the prior art, a preservation solution (hereinafter referred to as the "conventional preservation solution") for preserving and transporting various bacteria contained in human feces is described in Patent Document 3 (U.S. Patent Application Publication No. 2022 / 0160337). However, since the pH of this conventional preservation solution is set to 7.5, there are doubts about the preservation performance 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 in a modified GAM liquid medium having the following composition under anaerobic conditions at 37 °C for 72 to 96 hours.

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

[0145] (2) In addition, the composition of the bacterial preservation solution of the present invention was as follows and was designated as Sample 8. Bacterial preservation solution of the present invention (composition): ·L-cysteine hydrochloride... 1 g / L ·50% (w / v) glycerol... 500 mL ·D-PBS... 500 mL ·NaHCO3... as appropriate (added until pH 4.0 is reached)

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

[0147] (3) After culturing the Akkermansia, it was separated into a supernatant and an Akkermansia cell pellet by centrifugation, and the supernatant was removed. After removing the supernatant, the cell pellet was washed once with D-PBS, centrifuged, and then the supernatant was removed. The cell pellet was resuspended in 10 mL of D-PBS to prepare an Akkermansia suspension. This Akkermansia suspension was serially diluted with 10 -3 、10 -4 、10 -5 After that, 80 μL of these diluted solutions was added and spread on a modified GAM agar plate, and cultured under anaerobic conditions at 37 °C for 72 - 96 hours. After culturing, the colonies formed on the agar plate were counted, and the viable cell count of Akkermansia in the suspension was calculated.

[0148] (4) To 5 mL each of sample 8, which is the bacterial preservation solution of the present invention prepared in (2), and sample 9, which is the conventional preservation solution, 500 μL of the Akkermansia suspension in (3) was added and mixed, and stored at 4 °C for 5 days, 7 days, and 10 days. After storage, these mixtures were serially diluted with 10 -3 、10 -4 After that, 80 μL of each diluted solution was added and spread on a modified GAM agar plate, and cultured under anaerobic conditions at 37 °C for 72 - 96 hours. After culturing, the colonies formed on the agar plate were counted, and the viable cell count of Akkermansia in each mixture was calculated.

[0149] The ratio of the viable cell count cfu in (4) to the viable cell count cfu in (3) was defined as the survival rate of Akkermansia for each number of days elapsed. That is, the survival rate (%) = viable cell count cfu after storage at 4°C (5 days, 7 days, 10 days) × 9.09 / viable cell count cfu in 500 μL Akkermansia suspension × 100. The reason for multiplying by 9.09 is that 500 μL of Akkermansia suspension was added to 5 mL of the preservation solution, so the Akkermansia suspension was "diluted 9.09 times (0.5 mL / 5.5 mL × 100)".

[0150] Results The viable cell count cfu of sample 8, which is the bacterial preservation solution of the present invention, and sample 9 of the conventional preservation solution were 2.12E+07 at 0 day, 1.22E+07 for sample 8 and 3.49E+06 for sample 9 at 5 days, and 4.36E+06 for sample 8 and 1.90E+06 for sample 9 at 10 days. From this, the above survival rates were as follows. Sample 8... 100% (0 day), 57.4% (5 days), 20.6% (10 days) Sample 9... 100% (0 day), 16.5% (5 days), 8.9% (10 days)

[0151] Discussion From the results of the survival rate of Akkermansia, the bacterial preservation solution of the present invention in sample 8 had a higher viable cell count and survival rate of Akkermansia than the conventional preservation solution in sample 9. Considering the reason, the pH of the bacterial preservation solution of the present invention is adjusted to 4 on the acidic side to enhance the deoxygenation effect of L-cysteine hydrochloride, which is a reducing agent, while the pH of the conventional preservation solution is adjusted to 7.5 on the weakly alkaline side, and it is considered 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 during pH adjustment, and it is considered that this exhibits a synergistic effect of deoxygenation with L-cysteine hydrochloride.

[0152] Although Akkermansia is shown in Fig7D of Patent Document 3 (US Patent Application Publication No. 2022 / 0160337), this represents the abundance of Akkermansia OTU contained in Total OTUs (operational taxonomic unit: essential genes of bacteria (generally 16S ribosomal RNA gene)) in feces, showing the gene amounts of both live and dead bacteria combined. Therefore, it is unclear how much live Akkermansia is contained, and such descriptions in Patent Document 3 do not explain the survival of beneficial bacteria.

[0153] <Experiment 6: Preparation of Gel-like Bacterial Preservation Solution and Viscoelasticity Measurement> Preparation of Bacterial Preservation 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. On the other hand, L-cysteine hydrochloride was dissolved in D-PBS(-) and the sodium bicarbonate solution was added, and this was mixed with the previously prepared glycerin solution in which gellan gum was dissolved at a ratio of 1:1 (vol). Then, six types of bacterial preservation solutions 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), different gellan gum concentrations, and a pH of 4 were prepared. The gellan gum concentrations and sample numbers of these six types of bacterial preservation solutions are as follows. Sample 11 with a gellan gum concentration of 0.02% (w / v), Sample 12 with a gellan gum concentration of 0.075% (w / v), Sample 13 with a gellan gum concentration of 0.1% (w / v), Sample 14 with a gellan gum concentration of 0.3% (w / v), Sample 15 with a gellan gum concentration of 0.35% (w / v), and Sample 16 with a gellan gum concentration of 0.40% (w / v).

[0154] Visual Evaluation Sample 11, with the lowest gellan gum concentration of 0.02% (w / v), had a higher viscosity than the case without adding gellan gum, but it was liquid and did not gel. On the other hand, Samples 12 to 16 became gel-like and did not flow out even when the tubes containing the samples were tilted. Also, for the bacterial preservation solutions of Samples 12 to 15, when feces were put into the bacterial preservation solution, the feces were mixed with the bacterial preservation solution, whereas for the bacterial preservation solution of Sample 16, the feces did not completely mix with the bacterial preservation solution even when put in.

[0155] Viscoelasticity measurement (1) - Strain dispersion For the above-mentioned Sample 12 and Sample 15, a strain dispersion test (measuring the storage modulus (G′) and loss modulus (G″) within the range of strain (%) from 0 to 1000 at 30 °C and 1.0 Hz) was performed using a rheometer MCR101 (manufactured by Anton Paar). As a result, for Sample 12 with a gellan gum concentration of 0.075% (w / v), within the range of strain from 3.98% to 39.8%, 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. From this, it was determined that within the range of strain from 3.98% to 39.8%, it was in the linear region where the changes in both moduli were small and stable. On the other hand, for Sample 15 with a gellan gum concentration of 0.35% (w / v), within the range of strain from 0.0626% to 1.58%, 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. From this, it was determined that within the range of strain from 0.0626% to 1.58%, it was in the linear region where the changes in both moduli were small and stable.

[0156] Viscoelasticity measurement (2) - Frequency dispersion For the above-mentioned Sample 12 and Sample 15, based on the results of the strain dispersion test, strain (%) was selected from each linear region and frequency dispersion was measured. That is, in Sample 12, the frequency was changed between 0.6 and 62.8 Hz at 30°C with a strain (%) of 10%, and the changes in the storage modulus and loss modulus were measured. As a result, both the storage modulus and the loss modulus increased as the frequency increased. When the frequency exceeded 10.0 Hz, the storage modulus increased rapidly. Since the storage modulus > loss modulus at any frequency within the above range, Sample 12 is considered to be a gel-like substance from the results of the frequency dispersion test. In addition, in Sample 15, the frequency was changed between 0.6 and 62.8 Hz at 30°C with a strain (%) of 0.16%, and the changes in the storage modulus and loss modulus were measured. As a result, while the storage modulus increased as the frequency increased, the loss modulus showed a substantially constant value. Since the storage modulus > loss modulus at any frequency within the above range, Sample 15 is considered to be a gel-like substance from the results of the frequency dispersion test.

[0157] Viscoelastic measurement (3)……Temperature dispersion For the above-mentioned Sample 12 and Sample 15, based on the results of the strain dispersion test, the strain (%) was selected from each linear region, and the temperature dispersion was measured. That is, in Sample 12, the temperature was changed between 2.55 and 60°C at a frequency of 1.0 Hz with a strain (%) of 10%, and the changes in the storage modulus and loss modulus were measured. As a result, the storage modulus decreased as the temperature increased, showing a tendency to progress to a liquid (sol). Also, since the storage modulus > loss modulus at any temperature within the above range, Sample 12 is considered to be a gel-like substance from the results of the temperature dispersion test up to 2.55 to 60°C.

[0158] In addition, in Sample 15, the temperature was changed between 2.44 and 60.1°C at a frequency of 1.0 Hz with a strain (%) of 1%, and the changes in the storage modulus and loss modulus were measured. As a result, as the temperature increased, the storage modulus decreased, and the loss modulus started to increase after exceeding 35.5 °C. When the temperature reached 60 °C, the values of the storage modulus and the loss modulus became close. Therefore, if the temperature continues to rise beyond 60 °C, it is considered that the storage modulus < loss modulus. Also, at any temperature within the above range, the storage modulus > loss modulus. Thus, based on the results of the temperature sweep test, sample 15 is considered to be a gel substance from 2.44 to 60.1 °C.

[0159] Discussion on Viscoelasticity Test According to the results of the above viscoelasticity test, among the bacterial preservation solutions of samples 12 to 15 that are gel-like and mixed with feces, sample 12 with the lowest gellan gum concentration of 0.075% (w / v) had a linear region in the range of strain (%) 3.98 to 39.8, while sample 15 with the highest gellan gum concentration of 0.35% (w / v) had a linear region in the range of strain (%) 0.0626 to 1.58. Therefore, the linear region of the bacterial preservation solution with a gellan gum concentration between sample 12 and sample 15 is considered to be between strain (%) 0.063 and 39.8%. Thus, a bacterial preservation solution with strain (%) within this range is considered to be a suitable gel-like bacterial preservation solution that mixes with feces.

[0160] <Experiment 7: Preparation of Gel-like Bacterial Preservation Solution and Growth of Bifidobacterium> Test Purpose To examine the growth of Bifidobacterium in bacterial preservation solutions based on the difference in the content of the gelling agent [complex polysaccharide (Gellan gum)] in the bacterial preservation solutions.

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

[0162] Preparation of Bacterial Preservation Solution: Similar to Experiment 6 above, five types of bacterial preservation solutions with different gellan gum concentrations and a pH of 4 were prepared. The gellan gum concentrations and sample numbers of these five types of 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) (the 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) (the same as in Experiment 6).

[0163] On the other hand, about 2 g of feces collected from a human and 5 mL of sterilized water were mixed to prepare a fecal suspension. Then, 200 μL of this fecal suspension was added to 5 mL of the bacterial preservation solution of each sample and mixed. After that, it was stored at 4°C in the refrigerator for one week. After one week, a part was collected from this mixed solution, serially diluted 10-fold each time, spread on a plate medium, and anaerobically cultured at 37°C. Then, the colonies formed on the plate medium were counted to derive the viable count of Bifidobacterium in each mixed solution.

[0164] Results The viable count cfu of Bifidobacterium was 5.75E+06 in Sample 17, 4.375E+06 in Sample 18, 5.375E+06 in Sample 12, 5.75E+06 in Sample 19, and 5.75E+06 in Sample 20. In addition, in Sample 16, since the bacterial preservation solution became too hard and the addition and mixing of the fecal suspension did not go well, subsequent storage and culture were not performed. From the above, it can be seen that the change in the added amount of gellan gum did not cause a change in the viable count of Bifidobacterium, and there was no effect of gellan gum on the growth of Bifidobacterium. In addition, when observing the properties of the bacterial preservation solution of each sample, Sample 17 was a free-flowing liquid, while Samples 18, 12, 19, and 20 were gel-like and had excellent handleability. In addition, in Sample 16, as described above, it became too hard and the handleability also deteriorated. From the above, it was found that the gellan gum concentration is preferably 0.050 (w / v)% or more and 0.250 (w / v)% or less.

Explanation of Symbols

[0165] 10 Fecal collection kit 20 Inner container 21 Inner container body 22 Lid 23 Spoon-shaped spatula 24 Handle 25 Dimple 26 Baffle 27 Stirring ball 30 Outer container 31 Outer container body 32 Lid 33 Shielding film 40 Bacterial preservation solution

Claims

1. A method for preserving intestinal bacteria, comprising the steps of: The method includes an isolation step of isolating useful bacteria from the feces collected from a human being after a predetermined period of time has elapsed by mixing the feces collected from a human being with a predetermined bacterial preservation solution, The bacterial preservation solution contains a buffer solution selected from the group consisting of Dulbecco's phosphate buffered saline, phosphate buffered saline, HEPES buffer, and HEPES buffered saline, a reducing agent which is L-cysteine ​​hydrochloride, and a cryoprotectant which is glycerol, and has a pH adjusted to 3.5 to 5 with a pH adjuster selected from the group consisting of sodium bicarbonate and sodium hydroxide, and further contains 0.005 g to 0.02 g of the reducing agent relative to 10 mL in total of the buffer solution and the cryoprotectant.

2. A method for preserving intestinal bacteria, comprising the steps of: The method includes an isolation step of isolating useful bacteria from the feces collected from a human being after a predetermined period of time has elapsed by mixing the feces collected from a human being with a predetermined bacterial preservation solution, The bacterial preservation solution is a buffer solution of either Dulbecco's phosphate buffered saline or phosphate buffered saline, which contains a reducing agent which is L-cysteine ​​hydrochloride and a cryoprotectant which is glycerol, and the pH is adjusted to 3.5 to 5 with a pH adjuster which is sodium bicarbonate, and when the total amount of the buffer solution and the cryoprotectant is 10 mL, the method for preserving enterobacteria for preserving the useful bacteria further contains 0.005 g to 0.02 g of the reducing agent per 10 mL.

3. The method for preserving enterobacteria according to claim 1 or claim 2, wherein the bacterial preservation solution further contains a gelling agent, and the magnitude of strain (%) at which the storage modulus and loss modulus are in the linear region when strain is changed at 30°C and 1 Hz is 0.063 to 39.8%.

4. 4. The method for preserving intestinal bacteria according to claim 3, wherein the bacterial preservation solution contains 0.05 to 0.35% (w / v) of the gelling agent.

5. 3. The method for preserving intestinal bacteria according to claim 1, wherein the fecal collection liquid is one for which 10 to 14 days have passed since the feces was mixed into the bacterial preservation liquid.

6. The method further comprises a step of subjecting the feces collected from the human to a bacterial flora analysis step prior to the isolation step, 3. The method for preserving intestinal bacteria according to claim 1 or 2, wherein the isolation step isolates useful bacteria found to be present in the human in the bacterial flora analysis step.

7. The method for preserving intestinal bacteria according to claim 6, wherein in the bacterial flora analysis step, the obtained results of the intestinal flora are displayed by clearly indicating which type the sample belongs to among multiple types classified based on the type of intestinal bacteria present in abundance.

8. A bacterial preservation solution capable of isolating useful bacteria after a predetermined period of time by mixing feces collected from a human, A bacterial preservation solution comprising a buffer solution of any one of Dulbecco's phosphate buffered saline, phosphate buffered saline, HEPES buffer, or HEPES buffered saline, a reducing agent which is L-cysteine ​​hydrochloride, and a cryoprotectant which is glycerol, and having a pH adjusted to 3.5 to 5 with a pH adjuster of any one of sodium bicarbonate or sodium hydroxide, and further comprising 0.005 g or more and 0.02 g or less of the reducing agent per 10 mL of the total volume of the buffer solution and the cryoprotectant.

9. A bacterial preservation solution capable of isolating useful bacteria after a predetermined period of time by mixing feces collected from a human, A bacterial preservation solution comprising a buffer solution of either Dulbecco's phosphate buffered saline or phosphate buffered saline, a reducing agent which is L-cysteine ​​hydrochloride, and a cryoprotectant which is glycerol, and the pH is adjusted to 3.5 to 5 with a pH adjuster which is sodium bicarbonate, and further comprising 0.005 g or more and 0.02 g or less of the reducing agent per 10 mL of the total volume of the buffer solution and the cryoprotectant.

10. A bacterial preservation solution according to claim 8 or claim 9, further comprising a gelling agent, wherein the magnitude of strain (%) at which the storage modulus and loss modulus are in the linear region when strain is changed at 30°C and 1 Hz is 0.063 to 39.8%.

11. The bacterial preservation solution according to claim 10, wherein the content of the gelling agent is 0.05 to 0.35% (w / v).

12. 10. The bacterial preservation solution according to claim 8, wherein live cells of the beneficial bacteria can be obtained from the solution after 10 to 14 days have passed since the feces was mixed in.

13. A stool collection kit comprising a container containing the bacterial preservative solution according to claim 8 or 9, the bacterial preservative solution being present in an amount of 30 to 90 vol % of the content of the container.

14. A container for holding feces collected from a human; The bacterial preservation solution according to claim 8 or 9 contained in the container. Fecal collection kit.

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