JP2024044791A - Culture medium for intestinal bacteria and culture method for intestinal bacteria - Google Patents

Abstract

To provide a culture medium capable of culturing an expanded variety of intestinal bacteria, and a culture method.SOLUTION: The present invention provides a pre-culture medium for intestinal bacteria, usable for pre-culture prior to the main culture of human intestinal bacteria with the main culture medium. In the pre-culture medium for intestinal bacteria, mammal blood is added to a GAM medium. The mammal blood may be blood of at least one mammal selected from the group consisting of human, horse, and sheep. The volumetric concentration of the mammal blood may be 2.0% or more and 6.0%(v/v) or less.SELECTED DRAWING: Figure 1

Description

The present invention relates to a culture technique that can be commonly used for culturing a wide variety of enterobacteria, and particularly to a culture technique that makes it possible to simultaneously cultivate a variety of enterobacteria by modifying the medium used for preculture.

In recent years, it has become clear that intestinal bacteria have a significant impact on human health, and in order to elucidate their functions, bacterial flora analysis (analysis without culture) is being actively conducted, in which the DNA of intestinal bacteria in feces is directly decoded. However, because the function of half of the DNA base sequences obtained from bacterial flora analysis is unknown, it is difficult to elucidate the functions of all intestinal bacteria using bacterial flora, and there is a demand to culture intestinal bacteria and directly study and elucidate their functions.

In order to compare the phenotypes of many intestinal bacteria under equal conditions, a medium capable of culturing many kinds of bacteria at once is necessary. However, of the 56 most dominant species of intestinal flora in Westerners (see Table 1), 45 species have been successfully cultured by humans to date and are available from institutions that preserve and distribute strains, and similarly, of the 50 most dominant species of intestinal flora in Japanese people (see Table 2), 41 species are available, but the problem is that the culture medium recommended for each species by the distribution institution (see Tables 1 and 2) is specified by the distribution institution.

In other words, when culturing multiple types of enterobacteria simultaneously in a multi-well plate or the like, if a different recommended medium is used for each bacterium, it is necessary to prepare a different medium for each well, which creates the problem of taking a lot of time, effort, and cost. In addition, if the medium is different for each well, the culture medium in each well will become uneven due to the presence or absence of precipitate, etc., and there is a problem that it will be impossible to measure and compare the growth of bacteria in all wells at the same time.

The inventors therefore embarked on the development of a medium capable of culturing a large number of intestinal bacterial species without producing precipitates, and by culturing both pre-culture and main culture in Gifu University prescribed anaerobic medium (hereinafter referred to as "GAM medium"), they were able to culture 45 available species out of the 56 most dominant species of bacterial flora normally present in the intestines of Westerners, and were successful in culturing 32 of those species (see Non-Patent Document 1).

In addition, Non-Patent Document 2 reports that an improved version of GAM medium, mGAM medium with a light color and high transparency, was used to culture the 45 most dominant species of bacterial flora commonly found in the intestines of Europeans and Americans, and 34 species (76%) proliferated.

In addition, Non-Patent Document 3 reports that 70% of the genera detected in fecal samples can be isolated and cultured using a chemically defined medium called GMM, and Non-Patent Document 4 reports that 71% of the families can be isolated and cultured. Furthermore, Non-Patent Document 2 reports that 33 of the 45 most dominant species of bacterial flora commonly found in the intestines of Europeans and Americans (73%) can be cultured using the same medium.

Gotoh A, Nara M, Sugiyama Y, Sakanaka M, Yachi H, Kitakata A, et al. Use of Gifu Anaerobic Medium for culturing 32 dominant species of human gut microbes and its evaluation based on short-chain fatty acids fermentation profiles. Bioscience, Biotechnology, and Biochemistry (2017) 81:2009-17. doi: 10.1080/09168451.2017.1359486 Tramontano M, Andrejev S, Pruteanu M, Klunemann M, Kuhn M, Galardini M, et al. Nutritional preferences of human gut bacteria reveal their metabolic idiosyncrasies. Nature Microbiology (2018) 3:514-22. doi: 10.1038/s41564-018 -0123-9 Goodman AL, Kallstrom G, Faith JJ, Reyes A, Moore A, Dantas G, et al. Extensive personal human gut microbiota culture collections characterized and manipulated in gnotobiotic mice. Proceedings of the National Academy of Sciences (2011) 108:6252-7. doi: doi:10.1073/pnas.1102938108 Rettedal EA, Gumpert H, Sommer MOA. Cultivation-based multiplex phenotyping of human gut microbiota allows targeted recovery of previously uncultured bacteria. Nature Communications (2014) 5:4714. doi: 10.1038/ncomms5714

However, the culture methods disclosed in Non-Patent Documents 1 to 4 have a problem in that they are still unable to commonly culture a sufficient number of species of enterobacteria.
The present invention has been made in consideration of the above problems, and aims to provide a culture medium and a culture method capable of culturing a greater number of species of enterobacteria.

The invention made to solve the above problems is an intestinal bacteria preculture medium that can be used for preculture before main culture of human intestinal bacteria using a main culture medium, and which is a GAM-based medium. It is characterized by the addition of mammalian blood.

The enterobacteria preculture medium of the present invention is a GAM-based medium to which mammalian blood has been added, making it possible to simultaneously culture many types of enterobacteria.

The mammalian blood is preferably blood of at least one mammalian species selected from the group consisting of humans, horses, and sheep. This allows a greater variety of intestinal bacteria to be cultured.

Preferably, the mammalian blood is horse blood. In this way, even more species of intestinal bacteria can be cultured.

In the present invention, horse blood may be added to a GAM-based medium by mixing a GAM-based medium and an EG medium containing horse blood. By doing so, many species of intestinal bacteria can be cultured in the same manner as in a GAM-based medium to which horse blood is added.

The volume concentration of the mammal's blood is preferably 2.0% or more and 6.0% (v/v) or less. In this way, even more types of intestinal bacteria can be cultured.

The present invention provides a culture medium for enteric bacteria, comprising a preculture medium for preculturing enterobacteria, and a main culture medium for main culturing enterobacteria cultured in the preculture medium, which is a GAM-based culture medium. A main culture medium consisting of a preculture medium obtained by adding mammalian blood to a medium, and a GAM-based medium, an EGMB medium obtained by removing horse sterile defibrinated blood from an EG medium, or a mixed medium of a GAM-based medium and an EGMB medium. and an enteric bacteria culture medium.

The present invention includes a method for culturing intestinal bacteria, which is characterized by comprising a pre-culture step in which intestinal bacteria are pre-cultured using a GAM-based medium supplemented with mammalian blood, and a main culture step in which the intestinal bacteria cultured in the pre-culture step are cultured using a main culture medium.

In the intestinal bacteria culture method of the present invention, in the pre-culture step, intestinal bacteria with different pre-culture times are preferably inoculated into the multi-well a respective culture time before the start of the main culture step, with the intestinal bacteria with the longest culture time being inoculated into the multi-well in ascending order.
This saves pre-incubation time when culturing enterobacteria that require different incubation times.
It should be noted that the term "multi-well" used herein is not limited to a multi-well plate having many wells on one plate, but includes a multi-well plate in which many vials are arranged.

In addition, in the main culture step that follows, after transplanting the bacteria from the multiwell used in the preculture step to another multiwell all at once, the bacteria transplanted to the multiwell are main cultured for the same culture time. is preferred. In this way, a large number of intestinal bacteria can be cultured more efficiently.

Thus, according to the enterobacteria preculture medium, enterobacteria culture medium, and enterobacteria culture method of the present invention, many species of enterobacteria can be cultured simultaneously.

Regarding the culture tests of Example 1, Example 2, and Comparative Example 1, in which 45 species of the most dominant bacterial flora resident in Western human intestines were cultured in GAM medium, (A) is a flow diagram showing the test procedure. , (B) to (D) are each bacteria when GAM medium (Comparative Example 1), GE medium (Example 1), and GB medium supplemented with horse blood (Example 2) were used for preculture, respectively. It is a graph showing the degree of growth of. FIG. 1 is a portion of a graph showing the time course of growth of each bacterium in the culture test of Example 3, in which the 45 most dominant species of bacterial flora normally present in the intestines of Caucasians were pre-cultured in GB medium and then cultured in GAM medium. This is the rest of the same graph. FIG. 5 is a flow chart of the test procedure for Example 5, in which 11 species, excluding 26 species common to the 45 most dominant species of bacterial flora indigenous to the intestines of Westerners, from the 41 most dominant species of bacterial flora in Japanese people were pre-cultured in GB medium and then cultured in GAM medium. FIG. 5 is a graph showing the growth rate of each bacterium. Regarding the culture test of Example 5, in which 25 species of important intestinal bacteria were cultured using GB medium for preculture and GAM medium for main culture, and changing the culture time to 3 types, differences in growth due to differences in culture time were observed. This is a graph showing. FIG. 1A is a flow chart showing the test procedure, and FIG. 1B is a graph showing the growth rate of each bacterium, for Example 6, in which 51 species of indigenous intestinal bacteria were grown in GB medium supplemented with horse blood, pre-cultured in GB medium supplemented with sheep blood, and then main cultured in GAM medium. FIG. 13 is a flow chart showing the test procedure and FIG. 14 is a graph showing the growth rate of each bacterium in Example 7, in which 51 species of indigenous intestinal bacteria were grown in GB medium supplemented with horse blood, GB medium supplemented with human blood was used for preculture, and main culture was performed in GAM medium. FIG. 13 is a graph showing the growth of the 45 most dominant species of bacterial flora commonly found in the intestines of Caucasians for Example 8, in which GE medium, a 1:1 mixture of GAM medium and EG medium, was used for pre-culture, and EGMB medium, in which sterile defibrated horse blood was removed from EG medium, was used for main culture, and for Example 9, in which a mixture of EG medium and EGMB medium (EG+EGMB) was used for main culture.

Hereinafter, one embodiment of the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

(Intestinal bacteria preculture medium)
The intestinal bacteria preculture medium (hereinafter also simply referred to as "preculture medium") according to the present embodiment is used for preculture performed prior to main culture when culturing human intestinal bacteria. , is formed by adding mammalian blood to a GAM-based medium.

(GAM-based medium)
As the GAM-based medium used for forming the preculture medium, in addition to GAM liquid medium formed from GAM bouillon or modified GAM bouillon, GAM agar medium, modified GAM agar medium, and GAM semi-fluid high-layer medium (all of which can be manufactured by Nissui Pharmaceutical Co., Ltd.) can be appropriately used. As the GAM-based medium, GAM liquid medium formed from GAM bouillon or modified GAM bouillon is preferred because it is easy to handle on a multi-well plate, and GAM liquid medium formed from GAM bouillon is particularly preferred because it allows the cultivation of many enterobacteria.

(mixed medium)
As a method for adding mammalian blood to a GAM-based medium, there is a method of mixing a medium containing mammalian blood with a GAM-based medium. Examples of the culture medium to which mammalian blood is added by mixing with the GAM-based culture medium include EG medium, horse blood agar medium ( manufactured by Nissui Pharmaceutical Co., Ltd. ), and Brucella agar supplemented with horse defibrinated blood. A plate medium (made by Becton Dickinson & Company) can be mentioned, and EG medium is preferable because it contains horse blood and facilitates the addition of horse blood to GAM-based medium, and GAM-based medium This is preferable in that it is possible to culture a wide variety of intestinal bacteria by mixing with. The volume mixing ratio of the GAM-based medium and the EG medium (GAM-based medium/EG medium) is preferably 0.5 or more and 2.0 or less, particularly preferably 0.9 or more and 1.1 or less. Hereinafter, a mixed medium of GAM medium and EG medium will be referred to as GE medium. A mixed medium of "GAM-based medium" and "EG medium" shall be referred to as "GE-based medium."

(mammalian blood)
The mammalian blood to be added to the GAM-based medium is not particularly limited, and known mammalian blood such as human, horse, sheep, cow, rabbit, goat, donkey, dog, cat, etc. can be used as appropriate. Humans, horses, and sheep are preferable because many human intestinal resident bacteria can be cultured, and horse blood is particularly preferable because more intestinal intestinal bacteria can be cultured. Hereinafter, a medium obtained by adding horse blood to a GAM medium is referred to as a GB medium, a medium added to a sheep blood is referred to as a GB (sheep) medium, and a medium to which human blood is added is referred to as a GB (human) medium.

(Enterobacteria main culture medium (hereinafter simply referred to as "main culture medium"))
The medium used for this culture medium is not particularly limited, but may be a GAM-based medium, an EGMB medium obtained by removing sterile defibrated horse blood from EG medium, or a medium obtained by mixing a GAM-based medium and an EGMB medium. GAM-based medium, an EGMB medium obtained by removing sterile defibrated horse blood from EG medium, or a medium obtained by mixing a GAM-based medium and an EGMB medium are preferred in that they can commonly culture a wider variety of enterobacteria.
In addition, it is preferable to use GAM liquid medium for the main culture, since it is easy to prepare, has high transparency, and allows easy measurement of absorbance (OD ₆₀₀ ).

(Combined medium)
The combination medium is composed of a pre-culture medium and a main culture medium. The above-mentioned pre-culture medium and main culture medium can be appropriately combined to form a combination medium, but the combination of the pre-culture medium and the main culture medium is preferably any combination of GE medium and GAM-based medium, GB medium and GAM-based medium, GE medium and EGMB medium, or GE medium and (GAM-based medium + EGMB medium), and the combination of GB medium and GAM-based medium is particularly preferable.

(Intestinal bacteria culture method)
The intestinal bacteria culture method according to this embodiment includes a preculture step S1 and a main culture step S2. In this embodiment, various types of intestinal bacteria are simultaneously cultured.

(Preculture step S1)
The pre-culture step S1 is a culture step that is carried out prior to the main culture step S2 when culturing enterobacteria. In the pre-culture step S1, a GAM-based medium to which mammalian blood has been added is used as the pre-culture medium.

In the preculture step S1, enterobacteriaceae to be cultured are first transferred from a stored glycerol stock into a vial or multiwell plate into which a preculture medium has been dispensed in advance using a sterilized toothpick or other suitable method under anaerobic conditions. Port it with. The preculture medium is prepared before the start of the preculture step S1 and is kept in an anaerobic state.

The pre-culture time varies depending on the type of enterobacteria. Enterobacteria to be cultured are classified into groups that are pre-cultured for, for example, 24 hours, 48 hours, or 96 hours. For those that are pre-cultured for 96 hours, pre-culture is started 96 hours before the start of main culture, and for those that are pre-cultured for 48 hours and for those that are pre-cultured for 24 hours, culture is started 48 hours and 24 hours before the start of main culture, respectively. Each enterobacteria is cultured in a medium that allows easy visual confirmation of growth, such as a medium recommended by the distributor or a medium used for pre-culture made into an agar medium, and the degree of growth is visually observed at regular intervals to determine the culture time. The culture time is not limited to 24 hours, but can be divided into appropriate intervals, and is not limited to three groups, but may be divided into two or less, or four or more groups.

Here, it is preferable to transplant the enterobacteria with different culture times onto the same multi-well plate in sequence. Alternatively, the enterobacteria with different pre-culture times pre-cultured in vials or the like may be dispensed onto one multi-well plate. In this way, after pre-culture, the enterobacteria with different pre-culture times can be transplanted onto the multi-well plate for main culture at the same time using a copy plate.

(Main culture step S2)
The main culture step S2 is a step of culturing the intestinal bacteria cultured in the preculture step S1 in a main culture medium. A medium stored in advance under anaerobic conditions is dispensed into a multiwell plate or the like, and the intestinal bacteria cultured in the preculture step S1 are transplanted thereto and cultured.

The viability of the enterobacteria is determined, for example, by measuring OD ₆₀₀ using a spectrophotometer such as a Multiskan® GO microplate spectrophotometer (Thermo Fisher Scientific; product number 51119350).
Furthermore, whether the intestinal bacteria to be cultured have been cultured correctly can be confirmed by performing 16S rDNA sequence analysis using a known method.

Next, the present invention will be explained in further detail through a test using a culture medium according to an example of the present invention. However, the present invention is not limited to the following examples.

(Example 1)
Using GE medium, which is a mixture of GAM liquid medium and EG medium at a volume ratio of 1:1, as the pre-culture medium, and GAM liquid medium as the main culture medium, we collected the 45 most dominant bacterial flora that are available in Western human intestines. (see Table 1) was cultured.
The GAM liquid medium forming the GE medium used for preculture and the GAM liquid medium used for main culture were both prepared using GAM broth manufactured by Nissui Pharmaceutical Co., Ltd. according to the instructions.
EG medium was prepared according to the instructions of JCM (Japan Collection of Microorganisms). The pH of the EG medium was adjusted between pH 7.6 and 7.8 with NaOH. The composition of the EG medium is shown in Table 3.
The GE medium was formed by mixing 100 mL each of the GAM liquid medium and the EG medium in an anaerobic chamber, and the mixture was placed in an airtight container with an anero pack manufactured by Mitsubishi Gas Chemical Co., Ltd., and stored at 4°C.

The 45 most predominant strains of bacterial flora native to the intestines of Westerners were purchased from the distributors listed in Table 1. The "occupancy ranking" in Table 1 indicates the occupancy ranking in the intestines of Westerners.

(Preculture step S1)
Since the time required for pre-culture differs depending on the intestinal bacteria, the 45 most dominant species of indigenous bacterial flora in the intestines of Europeans and Americans were cultured on EG agar medium in advance, and the growth rate was observed every 24 hours to determine the pre-culture time for each intestinal bacteria (see Table 4). Based on this, pre-culture was started for each intestinal bacteria 24 hours, 48 hours, and 96 hours before the start of main culture.
First, a glycerol stock of bacteria that had been stored at -80°C and was capable of growing for 96 hours was transferred into an anaerobic chamber 96 hours before the start of the main culture, and the glycerol stock was scraped off with a sterilized toothpick and inoculated into 3.5 mL of GE medium that had been dispensed in advance into a 4 mL vial.
The GE medium in the vial and the glycerol stock of the added bacterial cells were mixed by inverting the vial, and the mixture was placed in a sealed container together with an Anaeropack and cultured at 37° C. for 96 hours.
Bacteria were inoculated and cultured in the same manner 48 hours and 24 hours before the start of the main culture.

(Preparation for main culture process)
Using a multichannel pipetman, reverse-spill 500 μL of the main culture GAM medium stored under anaerobic conditions into the wells of a 96-well deep well plate (round hole, round bottom, 1.0 mL; WATSON; product number 376-96R-10). It was dispensed by petting. At the same time, 1×PBS(-) was dispensed into one 96-well deep well plate, and used as a plate for cleaning the pin tips of Copy Plate 96 (Tokken Co., Ltd.; product number TK-CPST). These operations were performed in an anaerobic chamber.

(Main culture step S2)
500 μL of the preculture solution containing enteric bacteria cultured in the preculture step was once transferred to a 96-well deep well plate. Suspend the preculture solution in the 96-well plate by pipetting it 10 times with a multichannel pipetman, push the copy plate 96 into the preculture solution plate for 3 seconds, and push this into the main culture plate for 3 seconds. It was inoculated with. While washing the copy plate 96 with a pin tip washing plate, the preculture solution was inoculated into three 96-well plates for main culture. Gas Permeable Moisture Barrier Seal (4titude; product number, 4ti-0516/96) was applied to the inoculated 96-well plate, placed in a sealed container with Aneropack, and cultured at 37°C for 96 hours.

(Measurement of growth rate)
After the main culture process was completed, the growth rate of each enterobacteria was analyzed using _OD600 as an index. The results are shown in Figure 1. In the figure, the growth rate is shown as the average value obtained from three 96-well plates, and the error bars indicate the standard error.
The degree of growth (OD ₆₀₀ ) is the measured value of absorbance at 600 nm measured using a cuvette with an optical path length of 10 mm. In this embodiment, the measured value of absorbance of the culture solution at 600 nm was measured using a 96-well plate. Since the optical path length of this 96-well plate is different from 10 mm, the culture solution was placed in a cuvette (Sarsted Co., Ltd.; product number, 67.742J) and the absorbance was measured. The absorbance value of the cuvette/the absorbance value of the plate (hereinafter referred to as the "factor") was calculated for each enterobacteria. The value obtained when the culture solution was measured on the plate was multiplied by the factor to convert the value measured on the plate to the value measured on the cuvette.

(Example 2)
In the same manner as in Example 1, except that GB medium, which is a mixture of GAM medium and horse blood, was used as a preculture medium, the 45 most dominant species of Western human intestinal flora were cultured, and the cultured intestinal cells were cultured. The degree of growth ( _OD600 ) was measured. The results are shown in Figure 1.
For the GAM liquid medium used for the GB medium, put 11.8 g of GAM broth and Elix water in a 500 mL medium bottle to make 190 mL, stir with a stirrer bar to completely dissolve, and then autoclave (115 ° C., 15 minutes). As soon as the temperature inside the autoclave dropped to 97°C, it was placed in a sealed container with Anero Pack (Mitsubishi Gas Chemical Co., Ltd.; product number A-03) and stored at room temperature while being degassed. Horse defibrinated blood (Japan BioSealum) was dispensed in advance into Falcon tubes (CORNNG; product number 430791), placed in a sealed container with Aneropack, and subjected to anaerobic treatment for one day or more. Horse blood after anaerobic treatment was mixed with GAM medium in an anaerobic chamber to complete GB medium, which was placed in a sealed container with Aneropack and stored at 4°C until immediately before use. Table 5 shows the composition of the GB medium.

In Example 2, gDNA was extracted from the bacterial cells obtained from the preculture solution, and 16SrDNA sequence analysis was performed, followed by BLAST analysis of the results. As a result, the 16SrDNA sequences matched those of the target bacteria in the database, confirming that the growth of these bacteria was not the result of contamination.

(Comparative example 1)
In the same manner as in Example 1, except that GAM liquid medium was used for both the pre-culture medium and the main culture medium, the 45 most dominant bacterial flora resident in the Western human intestines were cultured, and the growth rate (OD) of the cultured intestinal cells was determined. ₆₀₀ ) was measured. The results are shown in Figure 1.

(Example 3)
In the same manner as in Example 2, 45 species of the most dominant bacterial flora resident in the Western human intestines were cultured using GB medium for preculture and GAM liquid medium for main culture, and the growth rate ( OD ₆₀₀ ) was measured to examine changes in growth over time during the main culture step. The results are shown in Figures 2-1 and 2-2. (Since each graph would be smaller if combined into one figure, it was divided into two figures.)
Furthermore, 16S rDNA analysis was performed using the preculture solution obtained upon completion of the preculture.

(Example 4)
As in Example 2, GB medium was used for pre-culture and GAM liquid medium was used for main culture to culture the 15 most dominant types of available Japanese intestinal flora (see Table 6). These 15 species are obtained by excluding 26 species out of the 41 most dominant intestinal flora species that overlap with the 45 most dominant intestinal flora species that are available. The preculture time for each enterobacterium was determined by inoculating each bacterium onto a GB plate medium in advance and visually observing the colonies formed every 24 hours (see Table 6). The main culture time was 96 hours in Example 2, but in this example, except for Eggerthella lenta, it was 48 hours, and for Eggerthella lenta, it was 96 hours. The degree of growth (OD ₆₀₀ ) of each cultured intestinal bacteria was measured, and 16S rDNA analysis was performed. The results are shown in FIG.

Example 5
As in Example 2, the important intestinal bacteria (including probiotic bacteria, butyric acid bacteria, and harmful bacteria) shown in Table 7 were cultured using GB medium for pre-culture and GAM liquid medium for main culture. The culture time was set to the following three times.
(1) Preculture using GB medium for 24 hours → Main culture using GAM medium for 24 hours (2) Preculture using GB medium for 24 hours → Main culture using GAM medium for 48 hours (3) Preculture using GB medium for 48 hours → Main culture using GAM medium for 24 hours

Example 9
The main culture medium was a mixture of GAM liquid medium and EGMB medium (GAM+EGMB), but the same procedure as in Example 8 was used to culture the 45 most predominant species of bacterial flora commonly found in the intestines of Europeans and Americans, and measure their growth rates.
To prepare the (GAM+EGMB) medium, 100 mL each of GAM medium (see Example 1) and EGMB medium (see Example 8) was mixed in an anaerobic chamber, placed in an airtight container together with an Anaeropack, and stored at room temperature.

(result)
(Results of Example 1, Example 2, and Comparative Example 1)
As shown in FIG. 1 , in Comparative Example 1 in which GAM medium was used for both pre-culture and main culture, only 36 of the 45 most dominant species of bacterial flora normally present in the intestines of Europeans and Americans (see the white circles marked with the symbol B in FIG. 1 ) grew, whereas in Example 1 in which EG medium was used for pre-culture, 40 of the 45 species grew sufficiently, and in Example 2 in which GB medium was used for pre-culture, 39 of the 45 species grew sufficiently (see the gray and black circles marked with the symbols C and D in FIG. 1 ).

(Results of Example 3)
As in Example 2, using GB medium as the pre-culture medium and GAM liquid medium as the main culture medium, the growth rate of bacteria during main culture was measured every 24 hours. As shown in Figure 2, among the 45 types of available Western intestinal bacteria, two of the bacteria reared in Example 2 did not grow, while one species did not grow in Example 2. It was confirmed that 39 bacterial species showed continuous growth. 16S rDNA analysis of the cultured bacteria confirmed that all 39 types of bacteria were growing reliably.

(Results of Example 4)
Using GB medium as the preculture medium and GAM liquid medium as the main culture medium, we cultured the 15 most dominant species of Japanese intestinal flora that were available, as shown in Figure 3. Although a sufficient growth rate (OD ₆₀₀ of 0.15 or more) was obtained for the seeds, it could not be confirmed by 16S rDNA analysis that the correct bacteria had been cultured for two of the seeds. Therefore, it was found that 12 of the 15 species could be cultured.

(Results of Example 5)
When 25 important enterobacteria species shown in Table 9 were cultured using GB medium for pre-culture and GAM liquid medium for main culture, sufficient growth was obtained for all 25 species (OD ₆₀₀ was 0.15 or more) and they were successfully cultured. There was no change in the number of culturable species compared to when pre-culture was performed in GAM medium, but the pre-culture time for three species was shortened from 48 hours to 24 hours (see Table 9).

(Results of Example 6)
GB (sheep) medium supplemented with 5% (v/v) sheep blood was used as the preculture medium, and GAM medium was used as the main culture medium. A culture test was performed on 51 species, including 39 species out of the 45 most dominant species of indigenous bacterial flora in Europeans and Americans that could be cultured in Example 3 and 12 species out of the 12 species out of the indigenous bacterial flora in Japanese people that could be cultured in Example 4, and as shown in Figure 5, sufficient growth (OD ₆₀₀ of 0.15 or more) was obtained for 48 species.

(Results of Example 7)
The same 51 types of intestinal bacteria as in Example 6 were cultured in the same manner as in Example 6, except that GB (human) medium supplemented with 5% (v/v) human blood was used as the preculture medium. When the test was conducted, as shown in FIG. 6, sufficient growth (OD ₆₀₀ of 0.15 or more) was obtained for 45 species.

(Results of Example 8 and Example 9)
Using EG medium for preculture and EGMB for main culture, we cultured the 45 most dominant species of Western human intestinal flora, and as shown in Figure 6, 39 species showed sufficient growth ( OD ₆₀₀ of 0.15 or higher) was obtained, and the 45 most dominant species of Western human intestinal flora were cultured using EG medium for preculture and (EGMB+GAM) medium for main culture. However, as shown in FIG. 6, sufficient growth (OD ₆₀₀ of 0.15 or more) was obtained for 40 species.

As described above, the medium of the present invention is not limited to the above embodiments, and for example, the main culture medium may be a medium other than GAM medium, EGMB medium, or a mixed medium of GAM medium and EGMB medium. The mammalian blood to be added to the GAM medium is not limited to horse, sheep, and human blood, and any known mammalian blood can be used as appropriate. Furthermore, blood from multiple types of mammals may be added at the same time. The volumetric concentration of mammalian blood added to the GAM medium may be less than 2.0% (v/v) or greater than 6.0% (v/v).

The invention made to solve the above problems is an enterobacteriaceae culture set medium consisting of a preculture medium and a main culture medium used for culturing human enterobacteria, the preculture medium being GAM. The medium consists of a GAM liquid medium formed from bouillon and horse, sheep, or human blood, and the volume concentration of the blood is 2.0% or more and 6.0% (v/v) or less; the culture medium consists of a GAM liquid medium formed from GAM broth;
Bacteroides uniformis, Parabacteroides merdae, Dorea longicatena, Ruminococcus bromii, Bacteroides caccae, Bacteroides thetaiotaomicron, Ruminococcus torques, Ruminococcus lactaris, Collinsella aerofaciens, Dorea formicigenerans, Roseburia intestinalis, Parabacteroides distasonis, Bacteroides ovatus, Eubacterium rectale, Bacteroides ides xylanisolvens, Coprococcus comes, Eubacterium ventriosum , Bacteroides dorei, Ruminococcus obeum, Subdoligranulum variabile, Pseudoflavonifractor capillosus, Holdemania filiformis, Bacteroides stercoris, Bacteroides eggerthii, Butyrivibrio crossotus, Bacteroides finegoldii, Parabacteroides johnsonii, Clostridium nexile, Anaerotruncus colihominis, Ruminococcus gnavus, Bacteroides intestinalis, Bacteroides fragilis, Clostridium asparagiforme, Enterococcus It is possible to simultaneously culture multiple bacteria including at least one type of bacteria selected from the group consisting of Faecalis, Clostridium scindens, and Blautia hansenii, Ruminococcus bromii, Eubacterium rectale, Subdoligranulum variabile, Holdemania filiformis, and Butyrivibrio crossotus. characterized by something.

The enterobacteria preculture medium of the present invention uses a preculture medium in which horse, sheep, or human blood has been added to a GAM-based medium at a volume concentration of 2.0% or more and 6.0% or less (v/v) , so that many types of enterobacteria can be cultured simultaneously.

The enteric bacteria culture medium of the present invention is preferably capable of simultaneously cultivating Ruminococcus bromii, Eubacterium rectale, Subdoligranulum variabile, Holdemania filiformis, and Butyrivibrio crossotus.

The enterobacteria culture medium of the present invention is selected from the group consisting of Bacteroides uniformis, Parabacteroides merdae, Dorea longicatena, Ruminococcus bromii, Bacteroides caccae, Bacteroides thetaiotaomicron, Ruminococcus torques, Ruminococcus lactaris, Collinsella aerofaciens, Dorea formicigenerans, Roseburia intestinalis, Parabacteroides distasonis, Bacteroides ovatus, Eubacterium rectale, Bacteroides xylanisolvens, Coprococcus comes, Eubacterium ventriosum, Bacteroides dorei, Ruminococcus obeum, Subdoligranulum variabile, Pseudoflavonifractor capillosus, Holdemania filiformis, Bacteroides stercoris, Bacteroides eggerthii, Butyrivibrio crossotus, Bacteroides finegoldii, Parabacteroides Preferably, the following bacteria can all be cultured simultaneously: Clostridium johnsonii, Clostridium nexile, Anaerotruncus colihominis, Ruminococcus gnavus, Bacteroides intestinalis, Bacteroides fragilis, Clostridium asparagiforme, Enterococcus faecalis, Clostridium scindens, and Blautia hansenii.

The present invention is a method for culturing enterobacteria, which involves culturing enterobacteria using an enterobacteria culture medium consisting of a pre-culture medium and a main culture medium, which is used for culturing human enterobacteria. The preculture medium consists of a GAM liquid medium formed from GAM broth and horse, sheep, or human blood added thereto, and the volume concentration of the blood is 2.0% or more, 6.0% (v /v) and the main culture medium consists of a GAM liquid medium formed from GAM broth,
Bacteroides uniformis, Parabacteroides merdae, Dorea longicatena, Ruminococcus bromii, Bacteroides caccae, Bacteroides thetaiotaomicron, Ruminococcus torques, Ruminococcus lactaris, Collinsella aerofaciens, Dorea formicigenerans, Roseburia intestinalis, Parabacteroides distasonis, Bacteroides ovatus, Eubacterium rectale, Bacteroides ides xylanisolvens, Coprococcus comes, Eubacterium ventriosum , Bacteroides dorei, Ruminococcus obeum, Subdoligranulum variabile, Pseudoflavonifractor capillosus, Holdemania filiformis, Bacteroides stercoris, Bacteroides eggerthii, Butyrivibrio crossotus, Bacteroides finegoldii, Parabacteroides johnsonii, Clostridium nexile, Anaerotruncus colihominis, Ruminococcus gnavus, Bacteroides intestinalis, Bacteroides fragilis, Clostridium asparagiforme, Enterococcus Cultivating simultaneously a plurality of bacteria including at least one type of bacteria selected from the group consisting of Ruminococcus bromii, Eubacterium rectale, Subdoligranulum variabile, Holdemania filiformis, and Butyrivibrio crossotus among the group consisting of Faecalis, Clostridium scindens, and Blautia hansenii. Including a method for culturing intestinal bacteria characterized by :

In the intestinal bacteria culture method of the present invention, it is preferable to simultaneously culture Ruminococcus bromii, Eubacterium rectale, Subdoligranulum variabile, Holdemania filiformis, and Butyrivibrio crossotus.

The intestinal bacteria culture method of the present invention includes Bacteroides uniformis, Parabacteroides merdae, Dorea longicatena, Ruminococcus bromii, Bacteroides caccae, Bacteroides thetaiotaomicron, Ruminococcus torques, Ruminococcus lactaris, Collinsella aerofaciens, Dorea formicigenerans, Roseburia intestinalis, Parabacteroides distasonis, Bacteroides ovatus, Eub acterium rectale, Bacteroides xylanisolvens, Coprococcus comes, Eubacterium ventriosum, Bacteroide dorei, Ruminococcus obeum, Subdoligranulum variabile, Pseudoflavonifractor capillosus, Holdemania filiformis, Bacteroides stercoris, Bacteroides eggerthii, Butyrivibrio crossotus, Bacteroides finegoldii, Parabacteroides johnsonii , Clostridium nexile, Anaerotruncus colihominis, Ruminococcus gnavus, Preferably, Bacteroides intestinalis, Bacteroides fragilis, Clostridium asparagiforme, Enterococcus Faecalis, Clostridium scindens, and Blautia hansenii are all cultured simultaneously.

Thus, according to the enterobacteria culture medium and the enterobacteria culture method of the present invention, many species of enterobacteria can be cultured simultaneously.

Hereinafter, Example 1, Example 8, and Example 9 will be read as Reference Example 1, Reference Example 8, and Reference Example 9, respectively.
(Example 1)
Using GE medium, which is a mixture of GAM liquid medium and EG medium at a volume ratio of 1:1, as the pre-culture medium, and GAM liquid medium as the main culture medium, we used the most dominant bacterial flora in the Western human intestines available.
Five species (see Table 1) were cultured.
The GAM liquid medium forming the GE medium used for preculture and the GAM liquid medium used for main culture were both prepared using GAM broth manufactured by Nissui Pharmaceutical Co., Ltd. according to the instructions.
EG medium is JCM (Japan Collection of Microorga)
nisms). The pH of the EG medium was adjusted between pH 7.6 and 7.8 with NaOH. The composition of the EG medium is shown in Table 3.
The GE medium was formed by mixing 100 mL each of GAM liquid medium and EG medium in an anaerobic chamber, placed in a sealed container together with Mitsubishi Gas Chemical Co., Ltd.'s Anero Pack, and stored at 4°C.

（実施例６）
前培地として、ＧＡＭ培地にヒツジの血液を５％（ｖ／ｖ）添加したＧＢ（ｓｈｅｅｐ）培地を用いて、入手可能な欧米人腸内常在細菌叢最優勢種と、入手可能な日本人腸内常在細菌について、培養試験を行った。本培養時間は、４８時間とした。測定した各腸内細菌の生育度（ＯＤ６００）を図５に示す。
（実施例７）
前培地として、ＧＡＭ培地にヒトの血液を５％（ｖ／ｖ）添加したＧＢ（ｈｕｍａｎ）培地を用いて、実施例６と同じ腸内細菌について実施例６と同様に培養試験を行って、培養した各腸内細菌の生育度（ＯＤ６００）を測定した。その結果を、図６に示す。
（実施例８）
前培養培地として実施例１で用いたＥＧ培地を用い、本培養培地としてＥＧ培地から馬無菌脱繊血を除去した表８に記載のＥＧＭＢ培地を用い、入手可能な欧米人腸内常在細菌叢最優勢４５種の培養を行った。

(Example 5)
As in Example 2, GB medium was used for pre-culture and GAM liquid medium was used for main culture, and the important intestinal bacteria shown in Table 7 (including probiotic bacteria, butyric acid bacteria, and bad bacteria) were cultured. ) was cultured. The culture time was set to the following three types.
(1) 24 hours of pre-culture using GB medium → 24 hours of main culture using GAM medium (2) 24 hours of pre-culture using GB medium → 48 hours of main culture using GAM medium (3) Main culture using GB medium 48 hours of preculture using → 24 hours of main culture using GAM medium

(Example 6)
As a pre-culture medium, GB (sheep) medium, which is a GAM medium supplemented with 5% (v/v) sheep blood, was used to prepare the most dominant species of Western human intestinal flora available and the available Japanese. Culture tests were conducted on resident intestinal bacteria. The main culture time was 48 hours. The measured growth degree (OD600) of each intestinal bacteria is shown in FIG.
(Example 7)
A culture test was conducted in the same manner as in Example 6 on the same intestinal bacteria as in Example 6, using GB (human) medium in which 5% (v/v) human blood was added to GAM medium as a pre-medium. The degree of growth (OD600) of each cultured intestinal bacteria was measured. The results are shown in FIG.
(Example 8)
The EG medium used in Example 1 was used as a pre-culture medium, and the EGMB medium listed in Table 8, obtained by removing horse sterile defibrinated blood from the EG medium, was used as the main culture medium. The 45 most dominant species were cultured.

(result)
(Results of Example 1, Example 2, and Comparative Example 1)
As shown in FIG. 1 , in Comparative Example 1, in which GAM medium was used for both pre-culture and main culture, only 36 of the 45 most dominant species of bacterial flora normally present in the intestines of Europeans and Americans (see the white circles marked with the symbol B in FIG. 1 ) grew, whereas in Example 1, in which GE medium was used for pre-culture, 40 of the 45 species grew sufficiently, and in Example 2, in which GB medium was used for pre-culture, 39 of the 45 species grew sufficiently (see the gray and black circles marked with the symbols C and D in FIG. 1 ).

(Results of Example 7)
Cultivation tests were carried out on the same 51 species of enterobacteria as in Example 6 in the same manner as in Example 6, except that GB (human) medium supplemented with 5% (v/v) human blood was used as the preculture medium. As a result, as shown in FIG. 6, sufficient growth (OD ₆₀₀ of 0.15 or more) was obtained for 45 species.

(Results of Example 8 and Example 9)
Using EG medium for preculture and EGMB for main culture, we cultured the 45 most dominant species of Western human intestinal flora, and as shown in Figure 7 , 39 species showed sufficient growth ( OD ₆₀₀ of 0.15 or higher) was obtained, and the 45 most dominant species of Western human intestinal flora were cultured using EG medium for preculture and (EGMB+GAM) medium for main culture. However, as shown in FIG. 6, sufficient growth (OD ₆₀₀ of 0.15 or more) was obtained for 40 species.

Claims (9)

An intestinal bacteria pre-culture medium that can be used for pre-culture before main culture of human intestinal bacteria using the main culture medium,
An enterobacteria pre-culture medium comprising a GAM-based medium to which mammalian blood has been added. The enterobacteria preculture medium according to claim 1, wherein the mammalian blood is the blood of at least one mammalian species selected from the group consisting of humans, horses, and sheep. The enterobacteria pre-culture medium according to claim 1, wherein the mammalian blood is horse blood. The enterobacteria preculture medium according to claim 1, characterized in that horse blood is added to the GAM-based medium by mixing the GAM-based medium with an EG medium containing horse blood. The intestinal bacteria preculture medium according to claim 1, wherein the volume concentration of the mammalian blood is 2.0% or more and 6.0% (v/v) or less. The enterobacteria preculture medium according to claim 1,
An enterobacteria culture medium comprising a GAM-based medium, an EGMB medium obtained by removing sterile defibrated horse blood from an EG medium, or an enterobacteria main culture medium consisting of a medium obtained by mixing a GAM-based medium and an EGMB medium. A method for culturing intestinal bacteria, comprising a pre-culture step of pre-culturing intestinal bacteria using a GAM-based medium containing added mammalian blood, and a main culture step of culturing the intestinal bacteria cultured in the pre-culture step using a main culture medium. The method for culturing intestinal bacteria according to claim 7, wherein in the pre-culture step, intestinal bacteria with different pre-culture times are inoculated into the multi-wells by the respective culture times before the start of the main culture step, and the intestinal bacteria with the longest culture time are inoculated into the multi-wells in order. 9. The intestinal bacteria culture according to claim 8, wherein the bacteria are transplanted all at once from the multiwell used in the preculture step to another multiwell plate, and then the bacteria transplanted to the multiwell are main cultured for the same culture time. Method.