KR20240167519A - Universal culture media for gut microbes - Google Patents

Abstract

The present invention relates to a culture medium for the proliferation of intestinal microorganisms, and more specifically, to a 'universal intestinal microorganism culture medium' for the comprehensive cultivation of a majority of intestinal microorganisms constituting the intestinal microbiome.
The 'universal intestinal microorganism culture medium' according to the present invention is characterized in that it additionally contains at least one additive selected from the group consisting of crop additives and blood additives in a basal medium for intestinal microorganism culture containing essential complex components.
Since the 'universal intestinal microorganism culture medium' of the present invention can be used to isolate and culture most of the intestinal microorganisms that make up the intestinal microbiome, it is useful for developing new intestinal microorganisms and innovative therapeutic agents using them.

Description

{UNIVERSAL CULTURE MEDIA FOR GUT MICROBES}

The present invention relates to a culture medium for the proliferation of intestinal microorganisms, and more specifically, to a ‘universal intestinal microorganism culture medium’ for the comprehensive cultivation of a majority of intestinal microorganisms constituting the intestinal microbiome.

The intestinal microbes, which number 100 trillion, more than 10 times the number of human somatic cells, account for the majority of the human microbes, and the number of species is estimated to be 2,000 to 4,000. As important companions of somatic cells, intestinal microbes have evolved to maximize their functions by closely exchanging signals and stimuli, and help humans quickly adapt to the external environment.

Current research on the gut microbiome, the sum of gut microbes, relies on metagenomic next-generation sequencing (mNGS) methods, and mNGS analysis is used to identify gut microbes that are thought to be related to human health and disease. In order to determine whether the discovered candidate gut microbes affect disease prevention and treatment, phenotypic improvement, etc., proof-of-concept research is necessary to prove the correlation between cause and effect through animal experiments that can satisfy Koch's postulates. Since proof-of-concept research is basically conducted by administering candidate gut microbes to laboratory animals, laboratory culture of the gut microbes to be tested is essential.

The most important factor in the laboratory cultivation of intestinal microorganisms is the culture medium. Most intestinal microorganisms grow very slowly in existing media over a period of 3 to 7 days or do not grow at all. As a result, the intestinal microorganisms that have been discovered are still at the stage of culture medium discovery, and the development of therapeutics using intestinal microorganisms has not progressed, and the elucidation of the mechanism of action of the intestinal microbiome is also at a standstill (Lau JT et al. Genome Med, 2016, 8:72; Browne HP et al. Nature, 2016, 53:543).

Currently, the most commonly used culture medium is YCFA (Tidjani Alou M et al. Clin Microbiol Rev, 2020, 34:(1):e00129; Browne, H. P. et al. Nature, 2016, 533:543-546). YCFA is a medium that supports the growth of the widest range of intestinal microorganisms, and a total of 137 species of intestinal microorganisms have been successfully cultured. However, considering that the number of intestinal microorganisms that make up the human intestinal microbiome is estimated to exceed several thousand species, YCFA medium has clear limitations (Chang Y et al. Front Microbiol, 2019, 10:2891).

In order to develop innovative new drugs based on the gut microbiome, it is essential to develop a universal culture medium that can overcome the limitations of the YCFA medium, and many attempts have been made to this end. However, there have been no successful cases of developing a culture medium for gut microbiota that can be used universally for culturing slow-growing or unculturable gut microbiota.

Korean Patent Publication No. 2023-0029769 discloses a microbial culture medium for reducing precipitation in a bacterial fermentation process, but this too was not suitable for culturing various types of intestinal microorganisms.

Accordingly, the inventors of the present invention have made efforts to solve the above problems and, as a result, have confirmed that when a crop additive, a blood additive, or a combination of these is added to a ‘basal media for culturing intestinal microorganisms,’ an ‘universal intestinal microorganism culture medium’ that promotes the growth of intestinal microorganisms in a general manner, including slow-growing or unculturable intestinal microorganisms, can be produced, thereby completing the present invention.

The purpose of the present invention is to provide a ‘universal intestinal microorganism culture medium’ capable of universally cultivating intestinal microorganisms.

In order to achieve the above purpose, the present invention provides an ‘intestinal microorganism universal culture medium’ characterized in that it further comprises at least one additive selected from the group consisting of crop additives and blood additives in a ‘basal media for intestinal microorganism culture’ containing a carbon source, a nitrogen source, and organic and inorganic salts in a complex manner.

In the present invention, the crop additive is characterized in that it is at least one selected from the group consisting of oatmeal, brown glutinous rice, spinach, mung bean, adlay, ginger, broccoli, bean sprout, philmuri sprout, cham namul, beetroot, hulled hempseed, orange peel, ginseng, licorice, morus bark, buckwheat, balloon flower, and green onion leaves.

In the present invention, the blood additive is characterized in that it is whole blood or blood component of a mammal selected from the group consisting of pigs, sheep, cows, and horses.

In the present invention, the ‘intestinal microbial universal culture medium’ is characterized by containing 0.1 to 53.5 wt% of ‘basal media for intestinal microbial culture’, 0.1 to 20 wt% of crop additive, and 0.1 to 20 wt% of blood additive.

In the present invention, it is characterized in that the essential complex component included in the ‘basal media for intestinal microorganism culture’ is at least one extract selected from the group consisting of beef extract and yeast extract.

In the present invention, the basic medium is characterized in that it further comprises (i) at least one nutritional additive selected from the group consisting of protein additives, carbohydrate additives, and fat additives; (ii) at least one inorganic additive selected from the group consisting of inorganic salts and trace element solutions; and (iii) at least one organic additive selected from the group consisting of urea, hemin, and vitamins.

Since the ‘universal intestinal microorganism culture medium’ of the present invention can be used to isolate and culture most of the intestinal microorganisms that make up the intestinal microbiome, it is useful for developing new intestinal microorganisms and innovative therapeutic agents using them.

Figure 1 shows the results of measuring the viable cell count (CFU/ml) of 10 types of intestinal microbial culture solutions cultured in the 'intestinal microbial universal culture medium' of the present invention, to which 19 types of crop additives were each added to the basic medium (B: basal media, 1: hulled hempseed, 2: orange peel, 3: ginseng, 4: licorice, 5: morus bark, 6: balloon flower, 7: buckwheat, 8: green onion leaf, 9: ginger, 10: bean sprout, 11: spinach, 12: philmuri sprout, 13: broccoli, 14: cham namul, 15: beet root), 16: mung bean, 17: adlay, 18: oatmeal, 19: brown rice.
Figure 2 shows the results of measuring the viable cell count (CFU/mL) of 10 types of intestinal microbial culture solutions cultured in the 'intestinal microbial universal culture medium' (hereinafter referred to as GM medium) of the present invention to which crop additives and blood additives are added to the basic medium (■: basic medium, □: GM medium).
FIG. 3 shows the results of comparing the number of intestinal microorganisms (CFU/gram) in the culture solution after culturing a human feces sample with a GM medium manufactured according to one embodiment of the present invention and a conventional YCFA medium (■: YCFA medium, □: GM medium).
Figure 4(4a-4b) shows the images obtained after 16-24 hours for the GM medium (left row) and 72 hours for the conventional YCFA medium (middle row) and the prescribed medium (right row) after inoculating and culturing seven species of low-growth, nutrient-poor intestinal microorganisms onto the existing YCFA medium, prescribed medium, and GM medium, respectively (1: Finegoldia magna , 2: Capnocytophaga granulosa , 3: Oribacterium asaccharolyticum , 4: Capnocytophaga ochracea , 5: Cutibacterium acnes , 6: Prevotella copri , 7: Porphyromonas gingivalis ).
Figure 5 shows a maximum likelihood phylogenetic tree obtained by DNA sequence analysis of the 16S rRNA gene of microorganisms cultured by inoculating 96 fecal samples of healthy adults on a GM medium. Figure 5a shows 422 known species with less than 99.80% sequence identity with the reference sequence, Figure 5b shows 1,027 unknown species with less than 99.80% sequence identity with the reference sequence, Figure 5c shows 56 unclassified species because their taxonomic system is not yet known, Figure 5d shows 55 gut fungal species, and Figure 5e is a tree of a total of 1,505 gut microbe library collections, where clades and inner circles are labeled by phylum and outer circles represent order levels.

The most important thing in laboratory cultivation of intestinal microorganisms is the culture medium. Most intestinal microorganisms grow very slowly in existing media over a period of 3 to 7 days (slow growth) or are unculturable, making isolation impossible.

The present inventors, taking into account the following facts: (a) in order to cultivate intestinal microorganisms in an in vitro environment, the medium must be composed to mimic the environment of gastrointestinal contents as much as possible; (b) in the intestines of humans who have lived an agricultural life for a long time, major metabolism occurs during the digestion and absorption of edible crops; and (c) the intestines are where ingested food is absorbed into the blood after digestion and where various metabolites of the blood are secreted, the inventors attempted to produce a universal culture medium for culturing intestinal microorganisms (Gut Microbiome, GM) by adding edible crops and blood components to a basic medium essential for microbial growth.

Accordingly, in the present invention, a ‘universal culture medium for gut microbes’ (universal medium or GM medium) was manufactured by adding a crop additive or a blood additive, or both, to a basal medium for culturing gut microbes containing essential complex components. As a result of evaluating the effect of using this medium on the culture of gut microbes, it was confirmed that it had the effect of promoting the culture of all types of gut microbes, including unculturable gut microbes.

Therefore, from one aspect, the present invention relates to a ‘universal intestinal microorganism culture medium’ characterized in that it further comprises at least one additive selected from the group consisting of crop additives and blood additives in a ‘basal media for intestinal microorganism culture’ containing essential complex components.

In the present invention, the crop additive is for promoting the growth of intestinal microorganisms, and may be exemplified by crops such as oatmeal, brown glutinous rice, spinach, mung bean, adlay, ginger, broccoli, bean sprouts, philmuri sprouts, cham namul, beetroot, hulled hempseed, orange peel, ginseng, licorice, morus bark, buckwheat, balloon flower, and green onion leaves, and a freeze-dried product or extract may be used in the form of powder, oil, or liquid.

When the above crops are applied in amounts of one or more, each crop may be freely mixed and applied without significant restrictions on the weight ratio, and preferably may be applied in a ratio of 1:1 to 5 (weight of one reference crop to weight of one of the remaining edible crops).

In the present invention, it is preferable that the crop additive be added in an amount of 0.1 to 20 wt% of the total composition of the ‘universal intestinal microorganism culture medium’. If the crop additive is less than 0.1 wt%, the effect of promoting the growth of intestinal microorganisms is minimal, and if it exceeds 20 wt%, there is a problem that the cost increases due to the increased amount added, but the culture efficiency does not increase significantly.

In the present invention, the blood additive is for promoting the proliferation of intestinal microorganisms and may be whole blood or blood components of mammals used as industrial animals. Examples of mammals include pigs, sheep, cows, horses, etc.

In the present invention, it is preferable that the blood additive be added in an amount of 0.1 to 20 wt% of the total composition of the ‘intestinal microorganism universal culture medium’. If the blood additive is less than 0.1 wt%, the effect of promoting the growth of intestinal microorganisms is minimal, and if it exceeds 20 wt%, there is a problem that the cost increases due to the increased amount added, but the culture efficiency does not increase significantly.

In the present invention, the basic medium for culturing intestinal microorganisms is a conventional medium used for culturing intestinal microorganisms and contains essential complex components.

In the present invention, the ‘basal media for intestinal microorganism culture’ may additionally include (i) one or more nutritional additives selected from the group consisting of protein additives, carbohydrate additives, and fat additives; (ii) one or more inorganic additives selected from the group consisting of inorganic salt and trace element solutions; and (iii) one or more organic additives selected from the group consisting of urea, hemin, and vitamins.

It is preferable that the above ‘basal media for intestinal microorganism culture’ be added at 0.1 to 53.5 wt% of the total composition of the ‘universal intestinal microorganism culture medium.’

In the present invention, the essential complex components may be exemplified by plant and animal extracts and microbial extracts that can be used for microbial culture, and it is preferable to use beef extract and yeast extract alone or together. It is preferable that the essential complex components are added in an amount of 0.1 wt% to 20 wt% in the ‘universal intestinal microbial culture medium’.

In the present invention, the nutritional additives may include one or more of protein additives, carbohydrate additives, and fat additives that can be used for microbial culture. It is preferable that the ‘intestinal microbial universal culture medium’ contains 0.1 to 10 wt% of protein additives, 0.1 to 10 wt% of carbohydrate additives, and 0.01 to 10 wt% of fat additives.

The above protein additive may include one or more amino acids and peptides available for microbial culture. Examples of amino acids include cystine, arginine, and glutamine. Examples of peptides include peptone compounds such as bacto-peptone, trypticase peptone, polypeptone, and tryptone, but are not limited thereto. It is preferable that the protein additive be included in the ‘universal intestinal microbial culture medium’ in an amount of 0.1 wt% to 10 wt%, 1 wt% to 10 wt%, 1 wt% to 5 wt%, or 1 wt% to 2 wt%.

In the present invention, the carbohydrate additive may include a single or two or more simple sugars or complex carbohydrates available for microbial culture, and examples thereof include, but are not limited to, glucose, sucrose, and soluble starch, and it is preferable that it be included in the ‘universal intestinal microbial culture medium’ in an amount of 0.1 wt% to 10 wt%, 1 wt% to 10 wt%, 1 wt% to 5 wt%, or 1 wt% to 2 wt%.

In the present invention, the fat additive may include one or more of short-chain fatty acids available for microbial culture (such as acetic acid, valeric acid, butyric acid, and furofionic acid), glycerol, and vegetable oils (such as soybean oil, corn oil, and canola oil), and is preferably included in the ‘universal intestinal microbial culture medium’ in an amount of 0.01 wt% to 10 wt%, 0.1 wt% to 10 wt%, 1 wt% to 10 wt%, 1 wt% to 5 wt%, or 1 wt% to 2 wt%.

In the present invention, the inorganic additive may include one or more inorganic salts and trace elements, and may be exemplified by Na ₂ HPO ₄ , NaCl, CaCO ₃ , C ₃ H ₃ NaO ₃ , trace elements (MnCl ₂ , FeSO ₄ , CoCl ₂ , ZnSO ₄ , Na ₂ MoO ₄ , NiCl ₂ , CuSO ₄ , H ₃ BO ₃ , MgSO ₄ , CaCl ₂ , (NH ₄ ) ₂ SO ₄ ), but is not limited thereto, and is preferably included in an amount of 0.001 wt% to 2.5 wt%, 0.001 wt% to 2 wt%, 0.001 wt% to 1.5 wt%, 0.001 wt% to 1 wt%, or 0.01 wt% to 1 wt% of the total medium.

In the present invention, the organic additive may include one or more of vitamins, urea, and hemin. The vitamins may include, but are not limited to, biotin, cyanocobalamin, folic acid, pyridoxine, riboflavin, L-ascorbic acid, coenzyme Q, inositol, niacin, dimethylglycine, L-carnitine, methylsulfonylmethane, and vitamin K1, and are preferably included in an amount of 0.00001 wt% to 1 wt% of the total medium.

The composition of the ‘universal intestinal microorganism culture medium’ according to the present invention is as follows.

furtherance Minimum
(weight%) Maximum
(weight%) (1) At least one essential complex component selected from the group consisting of plant, animal and microbial extracts (beef extract and yeast extract) 0.1 20 (1-i) One or more nutritional additives selected from the group consisting of protein additives, carbohydrate additives and fat additives; -Proteins (peptides and amino acids) 0.1 10 - Carbohydrates (simple sugars and complex carbohydrates) 0.1 10 -Fats (short-chain fatty acids, glycerol, and vegetable oils) 0.01 10 (1-ⅱ) One or more inorganic additives selected from the group consisting of inorganic salts and trace element solutions; 0.001 2.5 (1-ⅲ) One or more organic additives selected from the group consisting of urea, hemin and vitamins; 0.00001 1 (2) Blood additives 0.1 20 (3) Crop additives 0.1 20 (4) Water Remaining amount needed to reach 100%

The above-mentioned ‘universal intestinal microorganism culture medium’ dramatically promotes the growth of slow-growing intestinal microorganisms reported to date, and grows at a rate similar to that of generally known fast-growing microorganisms, and can form a single colony visualized on GM agar medium in just 12 to 18 hours, for example.

From another perspective, the present invention relates to a method for culturing intestinal microorganisms, comprising the steps of inoculating a culture or sample containing intestinal microorganisms into an ‘intestinal microorganism universal culture medium’; the step of culturing intestinal microorganisms; and the step of obtaining a microbial culture.

In the present invention, the intestinal microbial cells or sample used in the inoculation step refers to cells or samples containing intestinal microorganisms derived from the intestinal microbiome, and the sample may include various secretions (e.g., feces, urine, intestinal fluid, body fluid, blood), compositions, and cultures.

The culture may be the culture itself obtained by culturing intestinal microorganisms derived from the microbiome using the medium, or a culture supernatant obtained by removing the bacterial cells therefrom, or a concentrate or lyophilized product of the culture supernatant.

[Example]

Hereinafter, the present invention will be described in more detail through examples. It will be apparent to those skilled in the art that these examples are intended only to illustrate the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Preparation of ‘Intestinal Microbial Universal Culture Medium’ Containing Crop Additives

Not only humans who have lived an agricultural life, but also many mammals including primates have consumed various plant components through the metabolic process of microorganisms living in the intestines through evolution. The inventors who paid attention to this reported that the components cultivated and consumed by humans would be key components for the growth of intestinal microorganisms and attempted to confirm this. To this end, the inventors prepared 366 kinds of edible crops including commonly consumed vegetables, grains, and fruits, ground each one, freeze-dried it, and evaluated the effect on the culture of intestinal microorganisms.

First, in order to comprehensively cultivate the intestinal microorganisms of the present invention, the inventors attempted to create a medium that mimics the chemical composition of the human gastrointestinal tract as much as possible. To this end, a basal medium containing essential complex components, nutritional additives, organic additives, and inorganic additives was created as follows. First, 2.4 g of beef extract, 5.0 g of yeast extract, 10 g of bacto-peptone, 4.0 g of Na ₂ HPO ₄ , 1.5 g of glucose, 0.5 g of soluble starch, 1 g of L-cysteine-HCl, 2.0 g of trypticase peptone, 1 g of NaCl, 0.2 g of arginine, 0.2 g of sodium pyruvate, 1 g of urea, 0.2 g of CaCO ₃ , 2.0 g of polypeptone, 2 mL of glycerol, 0.1 mg of hemin, 1 mL of trace elements solution (0.72 gm/L MnCl ₂ x 4H ₂ O, 0.2 g/L _{FeSO 4 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 3 BO 3 ,} 0.6 _{g / L MgSO 4} 5mL butyric acid and 1mL (a mixture of iso-butyric acid) was mixed with distilled water to make 1 liter, and the final pH was adjusted to 7.4. The medium was sterilized, cooled to room temperature, and 2 mL of sterile vitamin solution (Biotin 0.02 g, Cyanocobalamin 0.02 g, P-aminobenzoic acid 0.03 g, Folic acid 0.05 g, Pyridoxine HCl 0.15 g, Thiamine HCl 0.05 g, Riboflavin 0.05 g, L-ascorbic acid 0.05 g, Coenzyme Q 0.04 g, L-glutamine 0.02 g, Trimethyl glycine 0.03 g, Inositol 0.03 g, Niacin 0.03 g, L-carnitine 0.03 g, Methylsulfonylmethane 0.03 g, Vitamin K1 0.1 mL to 1 L of water) was added. 200 μL of the following medium was added to each well of a 96-well plate and cultured in an anaerobic incubator for 3 hours to remove oxygen from the medium. 366 kinds of edible crop powders were prepared and added to each well at a concentration of 1.5 mg/mL.

To evaluate the cultivability, 10 common intestinal microorganisms ( Bacillus sp. strain AD39 SUBx, Citrobacter freundii strain CitB, Edwardsiella tarda strain KC-Pc-HB1, Staphylococcus gallinarum strain SSB39, Plesiomonas shigelloides strain MS-17-188, Clostridioides mangenotii strain CBA7501, Yersinia kristensenii Aeromonas sp. 1805, Escherichia coli strain 93-a pink, Lactococcus garvieae strain VX49 ) were prepared as pure cultures, 5 μL each was inoculated into each well, and the plates were cultured overnight in an anaerobic incubator at 37°C. By measuring OD ₆₀₀ the next day, the top 19 crops among 366 screened crops were identified as having excellent growth promotion efficacy for intestinal microorganisms.

In order to reconfirm the growth promotion efficacy by measuring the number of viable cells, the number of viable cells (Colony forming Unit, CFU) in the culture solution was measured to determine whether the growth of 10 types of intestinal microorganisms was promoted in the basic medium containing each of the top 19 crops, and the results are shown in Figure 1.

From Fig. 1, it was found that when oatmeal, brown glutinous rice, spinach, mung bean, adlay, ginger, broccoli, bean sprout, philmuri sprout, cham namul, beet root, hulled hempseed, orange peel, ginseng, licorice, mulberry bark, buckwheat, balloon flower, and green onion leaves were added as crop additives, the growth of intestinal microorganisms was significantly improved.

Next, in order to find out the appropriate addition level of crop additives, media were prepared by adding 0.1 wt%, 0.5 wt%, 2.5 wt%, or 10 wt% of an equal amount of a mixture of 19 crops to the basic medium containing only the essential complex components, beef extract (0.5 wt%) and yeast extract (1 wt%). 400 μL of each medium was added to a 48-well plate and cultured in an anaerobic incubator for 3 hours. Then, 10 μL of the 10 microbial cultures used in Example 1 were inoculated into each well and cultured in an anaerobic incubator at 37°C for 16 hours. The absorbance was measured and the relative increase is shown in Table 2.

Gut microbes Basic Badge 0.1 wt% 0.5 wt% 2.5 wt% 10% by weight Bacillus sp . + ++ ++ +++ +++ C. freundii + + + ++++ +++ E. tarda + + ++ ++++ +++ S. gallinarum + + + +++ +++ P. shigelloides + ++ ++ ++++ ++ C. mangenotii + + ++ ++++ +++ Y. kristensenii + + + ++++ +++ Aeromonas sp. + + + +++ ++ E. coli + ++ ++ ++++ +++ L. garvieae + + ++ +++ +++

* Absorbance increase rate of crop addition group compared to basic medium (essential complex components) control group: less than 10%: +, less than 50%: ++, less than 100%: +++, 100% or more: +++.

From Table 2, it was confirmed that when crop additives were added at 0.1% or more to the basic medium containing only beef extract and yeast extract as essential complex components without separately adding nutritional additives or inorganic additives, intestinal microorganisms with enhanced growth began to appear compared to the basic medium, and when added at 2.5% or more, an excellent growth promotion effect was observed in all tested microorganisms.

Example 2: Preparation of ‘Intestinal Microbial Universal Culture Medium’ Containing Blood Additives

The intestines are where ingested food is absorbed into the blood after digestion and where various metabolic products of the blood are secreted. The inventors, who paid attention to this, reported that blood components would be another key component that helps the growth of intestinal microorganisms, and to confirm this, they cultured intestinal microorganisms in a medium added with blood from various mammals (cows, pigs, sheep, horses). To this end, the basal media containing the essential complex components, nutritional additives, organic additives, and inorganic additives manufactured in Example 1 was sterilized and cooled, and only 3 wt% of the blood to be tested (whole blood) was added to prepare an ‘intestinal microorganism universal culture medium.’

400 μL of the manufactured ‘Intestinal Microbiota Universal Culture Medium’ was added to each well of a 48-well plate, and the medium was cultured for 3 hours in an anaerobic incubator to remove oxygen from the medium. 10 μL of the 10 types of microbial culture solutions used in Example 1 were inoculated into each well, and the plate was cultured for 16 hours in an anaerobic incubator at 37°C. The test group with the most prominent microbial growth was identified, and the results are shown in Table 3.

Gut microbes No blood Horse blood Sheep blood Pig blood Cow blood Bacillus sp . + + ++ ++ ++ C. freundii + ++ +++ +++ + E. tarda + + ++ +++ +++ S. gallinarum + ++ +++ +++ ++ P. shigelloides + +++ ++ ++++ ++ C. mangenotii + + ++ +++ ++ Y. kristensenii + ++ +++ ++++ ++ Aeromonas sp. + +++ +++ +++ +++ E. coli + ++ +++ ++++ ++ L. garvieae + ++ +++ ++++ ++

* Absorbance increase rate of blood-added group compared to basic medium (essential complex components + additives) control group: less than 10%: +, less than 50%: ++, less than 100%: +++, 100% or more: +++.

From Table 3, it was confirmed that the growth in the medium with added blood components was all improved compared to the basic medium when compared to the basic medium without blood components. In particular, excellent microbial growth was confirmed overall in the blood groups of sheep, pig, and cow, and the best growth was shown in pig blood among all 10 types of microorganisms, followed by sheep blood, cow blood, and horse blood.

Next, in order to find out the appropriate level of addition of blood additives, pig blood with excellent microbial culture efficacy was used to vary the addition level, and the remaining components were the same as in the experiment in Table 3 to prepare a basal media including essential complex components, nutritional additives, organic additives, and inorganic additives. 400 μL of each medium was added to a 48-well plate and anaerobically cultured for 3 hours. Then, 10 μL of the 10 types of microbial cultures used in Example 1 were inoculated into each well and cultured in an anaerobic incubator at 37°C for 24 hours. The absorbance was measured and the relative increase is shown in Table 4.

Inoculation bacteria 0.0 wt% 0.01 wt% 0.1 wt% 1 wt% 5 wt% 10% by weight Bacillus sp . + + + ++ +++ +++ C. freundii + + + ++ ++++ ++++ E. tarda + + + ++ ++++ ++++ S. gallinarum + + + ++ ++++ ++++ P. shigelloides + + ++ +++ ++++ +++ C. mangenotii + + ++ +++ ++++ +++ Y. kristensenii + + + +++ ++++ ++++ Aeromonas sp. + + + ++ ++++ ++++ E. coli + + + +++ ++++ +++ L. garvieae + + + ++ +++ +++

* Absorbance increase rate of blood-added group compared to basic medium (essential complex components + additives) control group: less than 10%: +, less than 50%: ++, less than 100%: +++, 100% or more: +++.

From Table 4, it was confirmed that when 0.1 wt% of blood additive was added, intestinal microorganisms with significantly improved growth began to appear compared to the control group without any additive, and when 5 wt% or more was added, it had an excellent growth promotion effect in all tested microorganisms.

Example 3: Preparation of ‘Intestinal Microbial Universal Culture Medium’ Containing Crop Additives and Blood Additives

Based on the results of Examples 1 and 2, the inventors of the present invention attempted to manufacture a universal medium for culturing intestinal microorganisms by adding 31 g of an equal amount of a mixture of 19 crops to the basal media composition including the essential complex components, nutritional additives, organic additives, and inorganic additives of Example 1, and adding distilled water to make a final volume of 950 mL. After sterilizing and cooling, 2 mL of a filter-sterilized (0.22 μm) vitamin solution and 50 mL of pig blood were added, and the final pH was adjusted to 7.4 to manufacture an ‘intestinal microorganism universal culture medium’ (hereinafter referred to as GM medium). 200 μL of each medium was added to a plate, and cultured in an anaerobic incubator for 3 hours. Next, 10 μL of the 10 types of microbial cultures used in Example 1 were inoculated into each well, and cultured in an anaerobic incubator at 37°C for 24 hours. Then, 100 μL of the original and diluted cultures were smeared to measure the CFU, which is shown in Figure 2.

From Fig. 2, it was found that the number of viable cells when culturing 10 types of intestinal microorganisms in the basic medium was in the range of 0.5 to 1 x 10 ⁹ CFU, whereas the number of viable cells in the GM medium with added crop additives and blood additives was in the range of 3.5 to 5 x 10 ⁹ CFU, indicating that the intestinal microorganism count increased by 7 to 10 times. In other words, when crop additives and blood additives were added to the basic medium, the culture of intestinal microorganisms was promoted.

Next, the intestinal microbial culturing ability of the GM medium was compared with that of the existing medium using human fecal samples. YCFA medium contains (per 1L of distilled water: Casitone 10g, Yeast Extract 2.5g, Glucose 2g, Sodium Bicarbonate 4g, Dipotassium Phosphate 0.45g, Monopotassium Phosphate 0.45g, Sodium Chloride 0.9g, Magnesium Sulfate Heptahydrate 0.01g, Calcium Chloride 0.09g, and Haemin 0.01g, L-cysteine 1g, 1 mL Resazurin (0.1%), 6.2 mL SCFA (a mixture of 17mL acetic acid, 6mL propionic acid, 1mL n-valeric acid, 1mL iso-valeric acid, 5mL butyric acid and 1mL iso-butyric acid), 2mL vitamin solution (water) Biotin 5mg, Vitamin B12 5mg, 1L After preparing p-Aminobanzoic acid (15 mg), Folic acid (25 mg), Pyridoxine HCl (75 mg), Thiamin HCl (25 mg), and Riboflavin (25 mg), it was anaerobically cultured in an anaerobic chamber at 37℃ to remove oxygen before use.

Figure 3 shows the results of comparing the viable cell counts (CFU) of the cultures after culturing the feces sample in the GM medium and the existing YCFA medium. Fresh feces samples collected from healthy human donors were immediately inoculated into the pre-anaerobic YCFA medium and GM medium to maintain the viability of the intestinal microorganisms. After culturing in an anaerobic chamber at 37℃, the viable cell counts (CFU) in the liquid culture were measured, and the intestinal microbial viable cell count obtained per feces sample was calculated as CFU/gram. The viable cell count when cultured in the existing YCFA medium was analyzed to be 0.57 x 10 ⁷ CFU/gram, whereas the intestinal microbial viable cell count obtained using the GM medium was analyzed to be 1.84 x 10 ⁷ CFU/gram. This means that the GM medium supports the growth of intestinal microorganisms 3.20±0.33 times more than the YCFA medium.

According to the results of Fig. 3, when a fecal sample is cultured using the GM medium of the present invention, the culture of intestinal microorganisms that constitute the intestinal microbiome is promoted, resulting in an effect of increasing the number of viable cells in the culture solution by more than three times compared to the YCFA medium.

Example 4: Evaluation of the efficacy of GM medium to promote intestinal microorganism growth

In Examples 1 to 3, it was confirmed that the ‘universal intestinal microorganism culture medium’ of the present invention is very effective in culturing intestinal microorganisms. Accordingly, slow growth intestinal microorganisms that are very difficult to culture and do not grow well and require culture for at least 72 hours even in the prescribed media recommended for each strain were subjected to a comparative analysis of the intestinal microorganism growth promotion efficacy according to each medium. The ‘universal intestinal microorganism culture medium’ (GM medium), YCFA medium, and prescribed media manufactured in Example 3 were prepared in the form of agar media, and then slow growth intestinal microorganisms were inoculated and cultured in an anaerobic incubator for up to 96 hours. Table 5 shows the tested slow growth intestinal microorganism taxonomy, deposit number, and prescribed media.

Slow Growth Microbes ID (Source) Prescribed medium Finegoldia magna GM Bank JBF6-00101 Columbia blood agar Cutibacterium acnes GM Bank JBP13-101 PYG Capnocytophaga ochracea JCM 12966 Columbia blood agar Oribacterium asaccharolyticum DSM 24638 Wilkins Chalgren anaerobic agar Prevotella copri JCM 13464 Schaedlar agar Porphyromonas gingivalis KCTC5352 p. gingivalis agar Capnocytophaga granulosa JCM 8564 capnocytophaga II agar medium

Figures 4a and 4b show images of seven low-growth intestinal microorganisms inoculated onto the existing YCFA medium, prescription medium, and ‘intestinal microorganism universal culture medium’ (GM medium), respectively, and the GM medium (left row) was imaged 16-24 hours later, while the existing YCFA medium (middle row) and prescription medium (right row) were imaged 72-96 hours later.

In the universal culture medium (GM medium) of the present invention, the growth of low-growth intestinal microorganisms was significantly promoted, and distinct culture colonies could be confirmed after 16 to 24 hours. On the other hand, both the YCFA medium and the prescription medium required at least 72 hours of culture time, and the culture conditions were not good.

According to the results of FIGS. 4a and 4b, it was found that the use of the GM medium of the present invention significantly promoted the growth of slow-growth intestinal microorganisms, significantly shortening the culture time until colony formation from 72 to 96 hours to less than 24 hours.

In addition to promoting the growth of various intestinal microorganisms, the GM medium of the present invention dramatically activated the growth of intestinal microorganisms that were hardly grown (Unculturable) or grew slowly (Slow Growth) in the culture medium, and were able to grow them as quickly as intestinal microorganisms that generally grow quickly. In other words, it can be seen that the GM medium of the present invention can provide universal growth of almost all intestinal microorganisms.

Example 5: Construction of a gut microbiota library using GM medium

It was confirmed that the GM medium prepared by adding crop additives and blood additives to the basic medium of the present invention as described in Examples 1 to 4 significantly promotes the growth of intestinal microorganisms compared to the existing medium. This means that the number of intestinal microorganisms that can be separated from the intestinal microbiome is significantly increased.

Herein, the inventors of the present invention constructed an intestinal microbial library using the GM medium of the present invention. To this end, 96 fresh fecal samples collected from healthy human donors were serially diluted using a pre-cultured GM liquid medium, spread on a pre-cultured GM agar medium, and cultured in an anaerobic chamber at 37°C. Microbial colonies were selected for further analysis after 1 day of culture. The genomic DNA of the intestinal microorganisms was isolated by homogenizing the samples with DNAzol (Invitrogen) and sonication according to the manufacturer's protocol, precipitating the DNA with ethanol, and then subjecting the samples to 16S rRNA sequence analysis. The full-length 16S rRNA gene was amplified by PCR using universal primers (SEQ ID NO: 1: 27F 5'-AGAGTTTGATCCTGGCTCAG-3' forward primer and SEQ ID NO: 2: 1492R 5'-GGTTACCTTGTTACGACTT-3' reverse primer) from the genomic DNA of the intestinal microorganisms. PCR conditions included denaturation at 95°C for 2 minutes, followed by 34 cycles of 95°C for 30 seconds, 52°C for 45 seconds, and 72°C for 2 minutes, and extension at 72°C for 5 minutes. The 16S rRNA gene purified from the gel slices was sequenced using the Sanger method, and the generated contigs were analyzed using a DNAbaser 4.36 analyzer to generate the DNA sequence of the full-length 16S rRNA gene. Then, the DNA sequence of the full-length 16S rRNA gene was subjected to taxonomic analysis based on sequence homology in NCBI BLAST, and a maximum likelihood phylogenetic tree was constructed using the ClustalW and MEGAX 45 programs to establish the taxonomic relationships among microbial strains.

Figures 5a to 5e are maximum likelihood phylogenetic trees of the intestinal microbial library constructed using the GM medium of the present invention. According to these, a total of 1,505 microorganisms, including 55 fungi and 1,450 bacteria, were isolated in the present invention. Of the 1,450 bacterial species, 392 were previously known species and were classified into 5 phyla, 15 classes, 29 orders, 59 families, and 115 genera, and 1,058 were previously unknown, unculturable species. In addition to the 16S rRNA base sequence, Gram staining and biochemical typing were performed on each identified microbial strain when necessary. All microorganisms, including 16S rRNA sequences and Gram stain images, were deposited in the Gut Microbe Bank (https://www.gmbank.org/) for public distribution. According to the results of FIG. 5, the present invention can provide a human intestinal microorganism library consisting of 1,505 types of microorganisms.

As examined in Examples 1 to 5, the GM medium of the present invention significantly promotes the growth of intestinal microorganisms compared to existing media (Figs. 1 to 3). That is, the GM medium of the present invention can universally support the growth of almost all intestinal microorganisms constituting the human gut microbiome.

In addition, the GM medium of the present invention can greatly promote the growth of slow-growing intestinal microorganisms (Fig. 4). Intestinal microorganisms known to be difficult to culture and grow very slowly generally take 3 to 7 days to observe single colonies in existing optimal media, but colonies are observed within 24 hours in the GM medium of the present invention (Fig. 4). This means that the GM medium of the present invention can be used as a universal medium for the cultivation of all intestinal microorganisms that constitute the human intestinal microbiome.

The present invention also provides the largest collection of intestinal microorganisms. One of the major differences between the present invention and the existing technology is that the collection of the present invention newly includes 1,027 new (unknown) intestinal microorganisms that were previously impossible to culture and thus were not isolated (Fig. 5).

Since the GM medium of the present invention universally supports most intestinal microorganisms, most intestinal microorganisms that were not identified because they could not be cultured in existing media were cultured in the present invention. The present invention collected 1,505 intestinal microorganisms from 96 individuals using the GM medium, and it is possible to collect up to several thousand species of intestinal microorganisms that constitute the human gut microbiome using the GM medium.

While the specific parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and that the scope of the present invention is not limited thereby. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

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Claims (6)

A 'universal intestinal microorganism culture medium' characterized in that it additionally contains at least one additive selected from the group consisting of crop additives and blood additives to the 'basal media for intestinal microorganism culture' containing essential complex components.
In the first paragraph, the crop additive is a 'universal intestinal microorganism culture medium' characterized in that at least one is selected from the group consisting of oatmeal, brown glutinous rice, spinach, mung bean, adlay, ginger, broccoli, bean sprout, philmuri sprout, cham namul, beetroot, hulled hempseed, orange peel, ginseng, licorice, morus bark, buckwheat, balloon flower, and green onion leaves.
'Intestinal microbial universal culture medium' characterized in that in the first paragraph, the blood additive is whole blood or blood components of a mammal selected from the group consisting of pigs, sheep, cows, and horses.
In claim 1, an 'intestinal microorganism universal culture medium' characterized by comprising 0.1 to 53.5 wt% of 'basal media for intestinal microorganism culture', 0.1 to 20 wt% of crop additive, and 0.1 to 20 wt% of blood additive.
In the first paragraph, the 'basal media for intestinal microorganism culture' is characterized in that the essential complex component included in the 'basal media for intestinal microorganism culture' is at least one extract selected from the group consisting of beef extract and yeast extract.
In claim 5, the 'basal media for intestinal microorganism culture' is characterized in that it further comprises (i) at least one nutritional additive selected from the group consisting of protein additives, carbohydrate additives, and fat additives; (ii) at least one inorganic additive selected from the group consisting of inorganic salt and trace element solutions; and (iii) at least one organic additive selected from the group consisting of urea, hemin, and vitamins.