Medium supplement for high yield cultivation of difficult-to-culture anaerobic microorganisms and medium composition containing the same

Abstract

To provide: a medium supplement and a medium composition which may produce anaerobes to be used as microbiome therapeutics or pharmabiotics in high yield in a stable manner over a long period of time; and a culture method for making it possible to economically culture fastidious anaerobes to a high final optical density.SOLUTION: A medium supplement for high-yield culture of anaerobes includes N-acetylhexosamine, L-aspartic acid, L-cysteine, and cobalamin. A medium composition for high-yield culture of fastidious anaerobes contains the medium supplement. A culture method is also provided.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a novel medium supplement for culturing difficult-to-cultivate anaerobic microorganisms at high concentrations, a medium composition containing the same, and a high-yield culture method for difficult-to-cultivate anaerobic microorganisms using the same. More specifically, the present invention relates to a medium supplement, medium composition, and culture method that enable mass production of difficult-to-cultivate anaerobic microorganisms including Akkermansia muciniphila and are suitable for use in pharmaceuticals and foods. [Background technology]

[0002] The human microbiome is a term that refers to the microbial community that lives symbiotically in the human body and the dielectric of such microbial community. It has become clear that the microbiome is closely correlated with human health, and has attracted much attention.

[0003] The rapid development of biotechnology research techniques, such as germ-free animal models, next-generation sequencing (NGS), and multi-omics analysis, has enabled us to not only analyze the composition and structure of intestinal microorganisms, but also to study the relationship between the function of the microbiome and diseases, leading to the publication of many research results. Microbiome therapeutics or permabiotic therapeutics (medical probiotics) have recently been attracting attention as they could be alternative treatments for infectious diseases, immune diseases, metabolic diseases, and other conditions that do not have effective treatments. If microbiome therapeutics or permabiotic therapeutics can be mass-produced and put into practical use, they are expected to be advantageously applicable to various intractable diseases.

[0004] Since the human intestine is in an anaerobic state, most of the microorganisms that make up the microbiome are anaerobic, but the reality is that most of these microorganisms are extremely limited in terms of available carbon and nitrogen sources, and are strictly anaerobic, meaning they are extremely sensitive to oxygen, making it difficult to culture them in large quantities and at high concentrations for industrial purposes. It is extremely difficult to culture strictly anaerobic microorganisms, and even more difficult to obtain a high biomass yield.

[0005] For example, A. muciniphila, which inhabits the mucous layer of the large intestine and is a promising candidate for permabiotics, may be cultured in media supplemented with hog gastric mucin as a carbon and nitrogen source (Derrien et al., 2004). A. muciniphila strains have also been cultured on Columbia broth (CB) and brain heart infusion (BHI) broth, but these media all contain animal-derived components and are less cultivable than most mucin-based media, and can only be grown to a final optical density that is about half that achieved with mucin-supplemented media.

[0006] Animal-derived ingredients are recognized as unsuitable for culturing anaerobic microorganisms for food and pharmaceutical applications in humans because they may contain contaminants of viral or bacterial origin, or may contain allergens, antigenic peptides or other undesirable products. Despite considerable efforts until recently, existing media for culturing anaerobic microorganisms are limited in that they are difficult to formulate, expensive, and cannot culture anaerobic microorganisms with high yields, making them unusable for industrial applications other than specialized research purposes. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention is intended to overcome the limitations or problems of the prior art described above, and one object of the present invention is to provide a medium supplement and medium composition that can be stably produced in high yields over a long period of time when anaerobic microorganisms used as microbiome therapeutics or permabiotics are industrially mass-cultured, and are suitable for use as pharmaceuticals, foods, or feeds.

[0008] Another object of the present invention is to provide a culture method that can economically cultivate fastidious anaerobic microorganisms to high final optical densities. [Means for solving the problem]

[0009] One aspect of the present invention for solving the above-mentioned problems relates to a medium supplement for high-yield culture of anaerobic microorganisms, comprising N-acetylhexosamine, L-aspartic acid, L-cysteine ​​and cobalamin.

[0010] The medium supplement of the present invention may contain N-acetylhexosamine 5 g / L, L-aspartic acid 8 g / L, L-cysteine ​​0.5 g / L, and cobalamin 0.0001 to 0.005 g / L.

[0011] The anaerobic microorganism may be a human intestinal obligate anaerobic microorganism, such as, but not limited to, Faecalibacterium prausnitzii, Anaerostipes caccae, Akkermansia muciniphila, Butyricicoccus pullicaecorum, Roseburia inulinivorans, Roseburia hominis, or Bifidobacterium longum.

[0012] Another aspect of the present invention for solving the above-mentioned problems is The present invention relates to a medium composition for high-yield cultivation of anaerobic microorganisms, which comprises a plant peptone, yeast extract, and dipotassium hydrogen phosphate, and further comprises fructose and lactose as carbon sources, and an amino acid mixture of N-acetylhexosamine, L-aspartic acid, and L-cysteine, and cobalamin as supplements.

[0013] The medium composition for high-yield cultivation of anaerobic microorganisms of the present invention may contain 2.5 g / L fructose, 2.5 g / L lactose, 20 g / L plant peptone, 10 g / L yeast extract, 2.5 g / L dipotassium hydrogen phosphate, 5 g / L N-acetylhexosamine, 8 g / L L-aspartic acid, 0.5 g / L L-cysteine, and 0.0001 to 0.005 g / L cobalamin.

[0014] The plant peptone may be selected from the group consisting of soy peptone, wheat peptone, cotton peptone, pea peptone, broadbean peptone, lupin peptone, and potato peptone, but is not necessarily limited thereto.

[0015] A further aspect of the present invention for solving the above-mentioned problems relates to a method for culturing anaerobic microorganisms in a high yield, which comprises inoculating the anaerobic microorganisms into the above-mentioned medium composition and growing them under anaerobic conditions.

[0016] The culturing step may be carried out under conditions of pH 6.6 to 7.0, culture temperature 35 to 39° C., stirring speed 40 to 50 rpm, nitrogen saturation 80 to 90%, hydrogen saturation 0 to 5%, and carbon dioxide saturation 5 to 20%.

[0017] In the high yield culture method of the present invention, the viable cell count of the cultured anaerobic microorganism is measured by the plate count method at 10 10It is characterized by reaching a cell density of ≥ CFU / mL. Effect of the Invention

[0018] According to various embodiments of the present invention, anaerobic microorganisms used as microbiome therapeutics or permbiotics can be stably produced in high yields when cultured industrially on a large scale, making them suitable for use as pharmaceuticals, foods, or feeds.

[0019] According to the medium supplement and medium composition of the present invention, Akkermansia muciniphila is a difficult-to-cultivate bacterial species that is a promising permabiotic candidate but is extremely sensitive to oxygen and dies even at trace amounts of oxygen, making it impossible to mass-produce it. However, this bacterial species can be cultured at high concentrations suitable for use in pharmaceuticals or foods.

[0020] The anaerobic microorganisms cultured in high yield in the medium supplement or culture medium of the present invention may be widely used in perbiotic medicines, lactic acid bacteria preparations, dairy products, probiotics, etc. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 is a schematic diagram showing the carbon metabolic process of an Akkermansia muciniphila strain. [Diagram 2] FIG. 1 is a schematic diagram showing the biosynthesis process of various amino acids from aspartic acid. [Diagram 3] 1 is a photograph showing the cell morphology of species-specific anaerobic microorganisms cultured in a medium containing the medium supplement of the present invention. [Figure 4] 1 is a photograph showing a change in the color of a medium over a culture time period in an incubator in which an Akkermansia muciniphila strain is cultured in an example of the present invention. [Diagram 5] The purity of the culture medium was confirmed through microscopic observation of Akkermansia muciniphila and PCR analysis using specific primers. [Figure 6]1 is a graph showing a growth curve (A) of an Akkermansia muciniphila strain and a change in pH (B) according to culture time in a medium of the present invention to which fructose has been added. [Figure 7] 1 is a graph showing a growth curve (A) of an Akkermansia muciniphila strain and a change in pH (B) according to culture time in the medium of the present invention to which maltose has been added. [Figure 8] 1 is a graph showing the growth curve (A) and pH change (B) of an Akkermansia muciniphila strain over culture time when cultured in the medium of the present invention supplemented with fructose using only high-purity nitrogen gas. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The present invention will now be described in detail with reference to the accompanying drawings.

[0023] In the present invention, the term "media" or "culture media" refers to a solid, semi-solid or liquid medium that contains all the nutrients and essential physical growth parameters required for the growth or proliferation of a microorganism.

[0024] As used herein, the terms "cultivation" or "growth" of a microbial organism means multiplication of the microbial organism in a given culture medium under conditions conducive to its growth.

[0025] As used herein, a "supplement" of a medium refers to an additive consisting of components selected to promote the growth, proliferation or other characteristics of a desired anaerobic microorganism.

[0026] As used herein, the term "anaerobic microorganisms" refers to microorganisms that are sensitive to oxygen and will not grow in the presence of oxygen. Anaerobic microorganisms may include strict or obligate anaerobic microorganisms and facultative anaerobic microorganisms.

[0027] As used herein, the terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Also, herein, recitations of numerical ranges by endpoints include all numbers within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, etc.).

[0028] One aspect of the present invention relates to a medium supplement for high-yield cultivation of anaerobic microorganisms, comprising N-acetylhexosamine, L-aspartic acid, L-cysteine ​​and cobalamin. The medium supplement may comprise 5 g / L N-acetylhexosamine, 8 g / L L-aspartic acid, 0.5 g / L L-cysteine ​​and 0.0001 g to 0.005 g / L cobalamin.

[0029] The medium supplement of the present invention is primarily used for culturing anaerobic microorganisms, such as gastrointestinal strict-anaerobic microorganisms in humans.

[0030] Examples of these anaerobic microorganisms include, but are not necessarily limited to, Faecalibacterium prausnitzii, Anaerostipes caccae, Akkermansia muciniphila, Butyricicoccus pullicaecorum, Roseburia inulinivorans, Roseburia hominis, or Bifidobacterium longum.

[0031] The medium supplement for high-yield culture of anaerobic microorganisms of the present invention contains N-acetylhexosamines. The N-acetylhexosamines may contain N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine (GalNAc), but preferably contains N-acetylglucosamine (GlcNAc). FIG. 1 is a schematic diagram showing the carbon metabolism process of Akkermansia muciniphila. Referring to FIG. 1, Akkermansia muciniphila has beta-galactosidase, an enzyme that converts lactose to galactose and glucose, and alpha-glucosidase, an enzyme that converts maltose to glucose, so that carbon sources such as lactose, maltose, and fructose are all used to form ATP, a high-energy molecule, through the corresponding process (glycolysis). N-acetylglucosamine (Glc-NAc) supplied from the outside is used for cell wall synthesis (peptidoglycan biosynthesis) and energy metabolism, and N-acetylglucosamine is metabolized to produce ammonia, which can also function to neutralize the cytoplasm and serve as a nitrogen source. In addition to N-acetylglucosamine (Glc-NAc), N-acetylgalactosamine (GaL-NAc) may also be added.

[0032] The N-acetylhexosamine may be included in an amount ranging from about 2.5 to 5 g / L.

[0033] Amino acids are important for maintaining the metabolic functions of cells cultured in cell culture media. In order to sustain good growth during high-density culture of anaerobic microorganisms, an external protein source is essential. The present inventors have confirmed that, among various amino acids, the combination of aspartic acid and cysteine ​​is highly effective as a protein source or nitrogen source for anaerobic microorganisms. The amino acids include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, and the mucin that Akkermansia muciniphila can decompose is characterized by a repeat structure of an amino acid sequence rich in serine, threonine, proline, and cysteine. However, in the present invention, among these amino acids, the addition of aspartic acid and cysteine ​​allows Akkermansia muciniphila to grow at a high yield. Aspartic acid and cysteine ​​may exist in the D-form and the L-form.

[0034] FIG. 2 is a schematic diagram showing the biosynthesis process of various amino acids from aspartic acid. Referring to FIG. 2, aspartic acid may be converted to homoserine, which is an intermediate in the biosynthesis of threonine and methionine, and various amino acids including serine and proline may be biosynthesized from aspartic acid. In addition, aspartic acid can improve resistance to acidic stress by increasing the production of pentose phosphates and nicotinamide adenine dinucleotide phosphate (NADPH), which are necessary for the synthesis of nucleic acids, fatty acids, and glutathione (a very important antioxidant in some bacteria). In addition, the biosynthesis of lysine and threonine from aspartic acid is useful for the proliferation of some bacterial species, and the intake of aspartic acid can increase the diversity of intestinal microbiota.

[0035] Media supplement compositions of the present invention may contain aspartic acid and cysteine ​​in amounts ranging from, for example, about 4-8 g / L and about 0.5-1 g / L, respectively.

[0036] The medium supplement of the present invention includes cobalamin, i.e., vitamin B12. Cobalamin is used by cells as a cofactor. If the culture medium does not include mucin, anaerobic microorganisms require a significant amount of cobalamin to grow to high density. Cobalamin may include compounds equivalent to cobalamin. For example, cobalamin may include cyanocobalamin, methylcobalamin, adenosylcobalamin, hydroxylcobalamin, and other functionally equivalent compounds. The culture medium supplement of the present invention may include cobalamin in an amount ranging from about 0.0001 to 0.005 g / L.

[0037] Vitamins include biotin, choline chloride, folic acid, myo-inositol, niacinamide, pyridoxine HCl, D-pantothenic acid (hemiCa), riboflavin, and thiamine HCl. Cobalamin alone increases the relative abundance of anaerobic microorganisms, and the addition of other vitamins has no significant effect on the biosynthesis of vitamin B12. Dielectric analysis of Akkermansia muciniphila showed that most strains, including ATCC BAA-835 and EB-AMDK19, have genes related to the biosynthesis of vitamin B12 (B1, B2, B3, B5, B6, B7, and B9), but not those related to the biosynthesis of vitamin B12. Cobalamin in Akkermansia muciniphila may also act as an important coenzyme for the synthesis of propionate from succinate.

[0038] Another aspect of the present invention relates to a medium composition for the high yield cultivation of anaerobic microorganisms comprising the medium supplement of the present invention.

[0039] As a basal medium for preparing a medium composition for high-yield cultivation of the anaerobic microorganism of the present invention, when the aim is industrial production by mass cultivation, a liquid medium is preferable, but a medium containing plant peptone, yeast extract, and dipotassium hydrogen phosphate may also be used.

[0040] The medium composition for culturing anaerobic microorganisms at high yields of the present invention is based on a medium containing plant peptone, yeast extract, and dipotassium hydrogen phosphate, contains fructose and lactose as carbon sources, and contains N-acetylhexosamine, an amino acid mixture of L-aspartic acid and L-cysteine, and cobalamin as supplements.

[0041] The medium composition of the present invention is suitable for culturing anaerobic microorganisms, specifically obligate anaerobic microorganisms. Non-limiting examples of obligate anaerobic microorganisms may include, but are not necessarily limited to, Faecalibacterium prausnitzii, Anaerostipes caccae, Akkermansia muciniphila, Butyricicoccus pullicaecorum, Roseburia inulinivorans, Roseburia hominis, or Bifidobacterium longum. The medium composition of the present invention is particularly suitable for culturing Akkermansia, specifically Akkermansia muciniphila, in high yield on an industrial scale.

[0042] The medium composition for high-yield cultivation of anaerobic microorganisms of the present invention may contain 20 g / L of plant peptone, 10 g / L of yeast extract, 2.5 g / L of dipotassium hydrogen phosphate, 2.5 g / L of fructose, 2.5 g / L of lactose, 5 g / L of N-acetylhexosamine, 8 g / L of L-aspartic acid, 0.5 g / L of L-cysteine, and 0.0001 to 0.005 g / L of cobalamin.

[0043] The medium for high-yield anaerobic microorganism culture of the present invention may contain a plant peptone. The plant peptone is a plant protein hydrolysate. It may be derived from any plant. The plant peptone may be selected from the group consisting of soy peptone, wheat peptone, cotton peptone, pea peptone, broadbean peptone, lupin peptone, and potato peptone. The plant peptone may be contained in an amount of, for example, about 15 to 20 g / L.

[0044] The medium for high-yield anaerobic microorganism culture of the present invention contains yeast extract. When yeast extract is added, an increase in the protein source can further increase the growth of anaerobic microorganisms on non-animal-derived media. The yeast extract may be yeast autolysate, ultrafiltered yeast extract or synthetic yeast extract. The concentration of the yeast extract may be 5 g / L to 10 g / L, for example, about 10 g / L.

[0045] The medium of the present invention may contain a phosphate-containing component, such as NaHPO 4、 The medium composition of the present invention further comprises K2HPO4 or KH2PO4. These components are added to the cell culture medium to maintain isotonic conditions and prevent osmotic imbalance. The medium composition of the present invention preferably maintains a pH in the range of 6.5 to 8.0, preferably 6.0 to 7.0, more preferably about pH 6.8±1.

[0046] The medium composition for high-yield cultivation of anaerobic microorganisms of the present invention contains fructose and lactose as carbon and energy sources, and may further contain maltose optionally. The concentration of fructose or maltose may be 2.5 g / L to 5.0 g / L, for example, about 2.5 g / L.

[0047] Although glucose may be included as a carbon source, the addition of glucose induces exponential growth of the microorganism, but this does not last long. Glucose can be supplied by decomposing lactose that is linked to galactose via a β(1→4)-glycoside, and since N-acetylglucosamine and glucose can be interconverted in the metabolic pathway, a combination of fructose and lactose is more preferable than glucose.

[0048] In the present invention, the culture medium is a "powdered medium" or "concentrated medium" containing multiple components, which may be provided in the form of a powder or concentrate as generally referred to, or may be combined with a volume of water to provide the desired concentration of a particular component in a liquid medium. Such powdered or concentrated media may be dissolved in an appropriate water, usually sterile water, before use.

[0049] In the present invention, media or media compositions include all final media containing components at concentrations suitable for culturing anaerobic microorganisms and suitable powders or concentrated media for dilution.

[0050] The culture medium of the present invention may optionally contain a reducing agent for culturing anaerobic microorganisms. A suitable reducing agent can promote the growth of anaerobic microorganisms by lowering the oxidation-reduction potential of the culture medium and scavenging dissolved oxygen (oxygen savanger). Suitable reducing agents include, but are not necessarily limited to, sodium thioglycolate, L-cysteine, dithiothreitol, dithioerythritol, sodium sulfide (NaS), and combinations thereof.

[0051] According to one embodiment of the present invention, the anaerobic nature of the medium of the present invention may be achieved by replacing oxygen in the medium with a mixed gas of nitrogen (N2), hydrogen (H2), and carbon dioxide (CO2) mixed in a volume ratio of 100:0:0 to 90:5:5. According to one embodiment of the present invention, the atmospheric pressure in the medium may be 0.1 to 0.3 atm, and preferably 0.2 atm (0.02 MPa).

[0052] Yet another aspect of the present invention relates to a method for culturing anaerobic microorganisms in a high yield, which comprises inoculating the anaerobic microorganisms into the medium composition of the present invention described above and growing them under anaerobic conditions.

[0053] The culture conditions of a microorganism can affect the growth rate of the microorganism, as is well known by those skilled in the art. In the present invention, the culture step may be performed at pH 6.6-7.0, culture temperature 35-39°C, stirring speed 40-50 rpm, nitrogen saturation 80-90%, hydrogen saturation 0-5%, and carbon dioxide saturation 5-20%.

[0054] In the present invention, the culture of anaerobic microorganisms was measured by optical density (OD) at a wavelength of 600 nm using a microplate reader. 600 ) until the cell density reaches 0.6 or more. At this point, the viable cell count measured by plate count method is 10 10 It can be cultured at high density to reach cell densities of > CFU / mL.

[0055] The culture temperature for high-concentration culture of anaerobic microorganisms is preferably 35 to 39°C, particularly 36 to 38°C. During culture, the composition inoculated with the microbial community suspension may be stirred. For example, the rotation speed (rpm) of the culture vessel may be 40 rpm to 50 rpm, but is not limited thereto. The culture period may be appropriately adjusted depending on the growth state of the anaerobic microorganisms, but is generally about 20 to 100 hours, particularly about 24 to 48 hours.

[0056] According to one embodiment of the present invention, the anaerobic nature of the medium of the present invention may be achieved by replacing oxygen in the medium with a mixed gas in which nitrogen (N2), hydrogen (H2), and carbon dioxide (CO2) are mixed in a volume ratio of 90:5:5 to 80:0:20. The replacement of oxygen in the medium with the mixed gas is preferably performed for 30 seconds or more based on 1 mL of medium volume, and most preferably for 2 minutes.

[0057] In A. muciniphila, hydrogen ions are used for ATP synthesis in the metabolic pathway, and hydrogen (H2) is important for anaerobic respiration by Ni-dependent hydrogenase under anaerobic conditions. Therefore, in order to culture A. muciniphila EB-AMDK19 strain at high concentration in the medium of the present invention, it is necessary to inject a mixed gas containing hydrogen or a gas containing carbon dioxide that dissolves in water to generate carbon dioxide and hydrogen ions.

[0058] The present invention will be described in more detail with reference to the following examples, but is not limited thereto. Unless otherwise specified, all parts and percentages referred to in the examples are by weight and all temperatures are expressed in degrees Celsius.

[0059] In the following examples, the concentration of anaerobic microorganisms (amount of biomass produced) was determined by measuring the optical density of the culture solution at 600 nm using a spectrophotometer during the culture period. ΔOD was calculated from the difference in absorbance between the initial period and after 48 hours (or 72 hours) of culture.

[0060] Working Example Example 1: Optimization of mucin-altering component composition in culture medium We explored the optimal combination of substrates and ingredients that could replace mucin based on soybean casein digestion liquid medium (Tryptic Soy Broth, TSB). To this end, Akkermansia muciniphila EB-AMDK19 (KCTC 13761BP) strain was inoculated into each prepared medium at a ratio of 0.1% v / v as shown in Table 1, and then cultured at 37°C under anaerobic conditions (90% N2, 5% CO2, 5% H2) for 24 to 48 hours, and the optical density (OD 600) was measured, and the results are shown in Table 1 below.

[0061] [Table 1]

[0062] Akkermansia muciniphila strains hardly grew in TSB medium, but it was found that they could be cultured by adding N-acetylglucosamine (GlcNAc) and threonine as alternatives to mucin. However, when threonine was replaced by aspartate, the culturability was improved at the same concentration (4g / L), and the culturability increased up to the addition of aspartate at a concentration of 8g / L.

[0063] As can be seen from Table 1, the change in absorbance in the medium containing N-acetylglucosamine, lactose, aspartic acid and vitamins based on TSB is at the level of 0.4, and the number of viable bacteria is 10 9 CFU / mL. In comparison, the change in absorbance in PM medium was at the 0.1 level, and the number of live bacteria at this time was 10 8 The results were measured as CFU / mL, showing a difference of more than 10-fold.

[0064] This is thought to be because the oxaloacetate / aspartate amino acid system is composed of lysine, asparagine, methionine, threonine and isoleucine, and aspartate can be converted to lysine, asparagine, methionine and threonine during metabolic processes.

[0065] Example 2: Comparison of the fertility of media containing different carbon sources To exclude animal-derived ingredients or ingredients produced using animal-derived enzymes, the media supplement of the present invention, based on plant peptones and yeast extract, was tested for anaerobic microbial culturability.

[0066] As a plant peptone, soybean peptone and yeast extract were tested in combination at concentrations of 5, 10, 15, and 20 g / L, respectively. As a result, the combination of soybean peptone at a concentration of 20 g / L and yeast extract at a concentration of 10 g / L showed the best culturing ability, and dipotassium hydrogen phosphate was added at a concentration of 2.5 g / L to adjust the pH, to prepare the basal medium.

[0067] To investigate the effect of carbon sources on the culturability of the anaerobic microorganism Akkermansia muciniphila, various carbon sources were added to the nitrogen source basal medium as shown in Table 2, and Akkermansia muciniphila EB-AMDK19 (KCTC 13761BP) strain was inoculated at a ratio of 0.1% v / v. The medium was then cultured at 37°C under anaerobic conditions (90% N2, 5% CO2, 5% H2) for 24 to 48 hours, after which the optical density (OD) was measured. 600 ) was measured, and the results are shown in Table 3 below.

[0068] [Table 2]

[0069] [Table 3]

[0070] As can be seen from the results in Table 3, the growth of Akkermansia muciniphila strains was much better when fructose was added than when lactose was used as the carbon source and N-acetylglucosamine was added, or when both N-acetylglucosamine and glucose were added. The change in absorbance (△OD) was at the average level of 0.5 to 0.6, and the viable cell count was about 10 10 The number of viable bacteria was measured as CFU / mL, and it was confirmed that the number of viable bacteria in the present invention increased by 10 to 100 times, and the culturability was significantly improved. In addition, when maltose, in which two glucose molecules are linked by a β(1→4)-glycoside, was added, the culturability was improved more than when glucose was added.

[0071] Example 3: Selection of important B vitamins for improving nutrient fertility In this example, we investigated whether the addition of B vitamins to the culture medium of Akkermansia muciniphila strains improved the growth rate. To select specific vitamins that affect the growth of anaerobic microorganisms, we tested all vitamins belonging to the B vitamin group individually and in the B vitamin complex (B1, B2, B3, B5, B6, B7, B9, B10, B12).

[0072] As shown in Table 4, each prepared medium was inoculated with Akkermansia muciniphila strain at a ratio of 0.1% v / v, and then incubated at 37°C under anaerobic conditions (90% N 2、 5% CO 2、 The plates were incubated in 5% H2 for 24-48 hours and the optical density (OD 600 ) was measured, and the results are shown in Table 4 below.

[0073] [Table 4]

[0074] As can be seen from Table 4, culturability increased only when cyanocobalamin (vitamin B12) was added to the medium (B12, B2+B6+B9+B12, B1+B2+B3+B5+B6+B7+B9+B12 or B1+B2+B3+B5+B6+B7+B9+B10+B12), and there was no significant difference when vitamin B12 was added alone compared to when vitamin B complex was added.

[0075] In order to find the optimal concentration of cobalamin, the optical density (OD) was measured after incubation with different concentrations of cobalamin. 600 ) was measured, and the results are shown in Table 5 below.

[0076] [Table 5]

[0077] As can be seen from the results in Table 5, the optimum concentration of vitamin B12 to be added to the medium was confirmed, and it was found that at concentrations below 0.1 mg / L, the culturability gradually decreased, and there was no significant difference in the culturability between concentrations of 0.1 mg / L and 5 mg / L, so it is believed that a concentration of 0.1 mg / L is most suitable.

[0078] Example 4: Optimization of medium components In order to explore the essential components and optimal combinations required for high-concentration cultivation of anaerobic microorganisms, Akkermansia muciniphila strains were cultivated under the same conditions in media containing various combinations of components, and the results are shown in Table 6 below.

[0079] [Table 6]

[0080] As a result of confirming the effect of each component on the culture in the medium of the present invention, it was confirmed that N-acetylglucosamine, a nitrogen-containing derivative of monosaccharide, is the most essential component for A. muciniphila culture, and that fructose and lactose, carbon sources that share part of the metabolic pathway with N-acetylglucosamine, are necessary components for high-concentration culture. The combination of soybean peptone, the main amino acid source, with aspartic acid and cyanocobalamin was confirmed to be important components for improving culturing ability.

[0081] Maltose can replace glucose and fructose, and the growth rate in the initial stage of cultivation is faster than that of fructose and glucose, and there is no significant difference in cultivability. However, since maltose is currently somewhat more expensive than fructose and glucose, fructose is considered to be the most suitable medium for industrial use. In conclusion, as can be seen from Table 6, all the components of the medium of the present invention are necessary for high-concentration cultivation of Akkermansia muciniphila strains, and it was confirmed that the best cultivability was achieved when all the components were combined.

[0082] Example 5: Cultivation confirmation of various Akkermansia muciniphila strains in the medium of the present invention The same procedure as in Example 1 was carried out using the medium shown in Table 2, except that the type of the cultured strain was changed as shown in Table 7 below. Akkermansia muciniphila strain was inoculated at a ratio of 0.1% v / v, and cultured at 37°C under anaerobic conditions (90% N2, 5% CO2, 5% H2) for 24 to 48 hours, and the optical density (OD 600 ) was measured and the results are shown in Table 7 below.

[0083] [Table 7]

[0084] As can be seen from the results in Table 7, the culture medium of the present invention was used to grow the Akkermansia muciniphila standard strain (ATCC BAA-835 T ) and the cultivability of different Akkermansia muciniphila strains were confirmed. As a result, all Akkermansia muciniphila strains showed similar cultivability levels, and therefore it can be confirmed that the medium can be used as a high-concentration culture medium for all Akkermansia muciniphila strains.

[0085] Example 6: Comparison of the growth rates of various difficult-to-culture strictly anaerobic microorganisms The same procedure as in Example 1 was carried out using the medium described in Table 2, except that the types of cultured strains were changed as shown in Table 8 below. Faecalibacterium prausnitzii, Anaerostipes caccae, or Bifidobacterium longum strains were inoculated at a ratio of 0.1% v / v, and cultured at 37°C under anaerobic conditions (90% N2, 5% CO2, 5% H2) for 24 to 48 hours, and the optical density (OD 600) was measured, and the results are shown in Table 8 below. For comparison, the bacteria were also cultured in a control medium known to have excellent culturability, and the culturability was compared with that in the medium of the present invention. As a control medium, F. Prausnitzii was cultured under the same conditions in a Brain Heart Infusion (BHI) medium supplemented with 5 g / L yeast extract, 1 g / L cellobiose, and 1 g / L maltose. A. caccae was cultured in a TSB medium supplemented with 5 g / L yeast extract, and B. longum was cultured in a BL broth medium.

[0086] [Table 8]

[0087] As can be seen from the results in Table 8, the feasibility of culturing difficult-to-culture obligate anaerobic microorganisms such as Faecalibacterium prausnitzii and Anaerostipes caccae, and Bifidobacterium longum was examined, and it was confirmed that the medium of the present invention has better culturability than the control medium known to have good culturability.

[0088] Example 7: Optimization of process conditions for mass cultivation - Comparative analysis of fertility according to cultivation conditions Using the anaerobic fermentation system shown in Figure 4, the medium in Table 2 was used to change the type of carbon source, and the culture was performed at a 3L scale under the conditions shown in Table 9 below, and the growth curve and pH change of the Akkermansia muciniphila strain were confirmed. In addition, the changes in the number of bacteria and the morphological changes were confirmed by microscopic observation and shown in Figure 5, and the viable bacteria number was measured using the plate count method and shown in Table 10.

[0089] [Table 9]

[0090] [Table 10]

[0091] As a result of culturing Akkermansia muciniphila EB-AMDK19 strain in an anaerobic fermentation system using the medium of the present invention, it was found that the exponential growth phase was 5 to 25 hours after inoculation, and a rapid change in pH was also confirmed to have occurred 9 to 18.5 hours after inoculation. The optical density (OD 600 ) is found to be at the 0.798 level, and the number of live bacteria at this time is 5.5x10 10 The CFU / mL were measured (see FIG. 6 and Table 10).

[0092] When fructose was replaced with maltose in the medium of the present invention, the exponential growth phase was found to be 3.5 to 21 hours after inoculation, and it was confirmed that the pH change occurred rapidly at 7 to 14 hours. Compared with the results of culturing using fructose, the medium of the present invention containing maltose showed a characteristic that the induction phase was shorter by 1.5 hours, and the absorbance was at the 0.632 level based on the 24-hour culture, which was somewhat lower, but the viable cell count at this time was 5.1 x 10 10 There was no significant difference from CFU / mL (see FIG. 7 and Table 9).

[0093] In the medium of the present invention containing fructose, cultivation was carried out using a more economical and industrially suitable hydrogen-free gas {high-purity nitrogen gas (100% N2) or a mixed gas composed of 80% N2 and 20% CO2} instead of a mixed gas (90% N2, 5% CO2, 5% H2). As a result, it was found that the culturing ability was significantly inferior to the conventional cultivation results in the case of high-purity nitrogen gas, but there was no significant difference in the culturing ability in the case of a gas composed of 80% N2 and 20% CO2 (see FIG. 8 and Table 10).

[0094] In addition, as can be seen from Table 10, all of the components in the medium of the present invention are necessary for high-concentration cultivation of Akkermansia muciniphila strains, and it was confirmed that injection of a mixed gas containing hydrogen and carbon dioxide is further necessary.

Claims

1. A medium composition for culturing anaerobic microorganisms, comprising 15 g / L to 20 g / L of plant peptone, 5 g / L to 10 g / L of yeast extract, and dipotassium hydrogen phosphate, the medium containing, as a carbon source, 2.5 g / L to 5.0 g / L of lactose and at least one of fructose and maltose, 2.5 g / L to 5 g / L of N-acetylhexosamine, 4 g / L to 8 g / L of an amino acid mixture of L-aspartic acid and 0.5 g / L to 1 g / L of L-cysteine, and 0.0001 g / L to 0.005 g / L of cobalamin, the N-acetylhexosamine is N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine (GalNAc); The medium composition for culturing an anaerobic microorganism, wherein the anaerobic microorganism is Anaerostipes caccae or Bifidobacterium longum.

2. The medium composition for culturing anaerobic microorganisms according to claim 1, characterized in that it contains 20 g / L of plant peptone, 10 g / L of yeast extract, 2.5 g / L of dipotassium hydrogen phosphate, 2.5 g / L of fructose, 2.5 g / L of lactose, 5 g / L of N-acetylhexosamine, 8 g / L of L-aspartic acid, 0.5 g / L of L-cysteine, and 0.0001 to 0.005 g / L of cobalamin.

3. 2. The medium composition for culturing anaerobic microorganisms according to claim 1, wherein the plant peptone is selected from the group consisting of soy peptone, wheat peptone, cotton peptone, pea peptone, broadbean peptone, lupin peptone, and potato peptone.

4. A method for culturing anaerobic microorganisms, comprising inoculating the medium composition according to claim 1 with anaerobic microorganisms and culturing the microorganisms under anaerobic conditions.

5. 5. The method for culturing anaerobic microorganisms according to claim 4, wherein the culturing step is performed under conditions of pH 6.6-7.0, culture temperature 35-39° C., stirring speed 40-50 rpm, nitrogen saturation 80-90%, carbon dioxide saturation 5-20%, and hydrogen saturation 0-5%.

6. The viable count of cultured anaerobic microorganisms was measured by plate count method. １０ The method for culturing anaerobic microorganisms according to claim 4, characterized in that a cell density of CFU / mL or more is reached.

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