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

Abstract

[Solution] The present invention relates to a medium supplement for the high-yield cultivation of anaerobic microorganisms, which contains N-acetylhexosamine, L-aspartic acid, L-cysteine, and cobalamin, and a medium composition for the high-yield cultivation of difficult-to-cultivate anaerobic microorganisms, and a cultivation method therefor.The present invention enables the high-concentration cultivation of anaerobic microorganisms, which are very difficult to cultivate for high yields, and provides an innovative method for the cost-effective mass cultivation of difficult-to-cultivate anaerobic microorganisms, which are particularly suitable for use in food and pharmaceutical applications.

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 in symbiosis with the human body and the dielectrics of such microbial communities. It has become clear that the microbiome is closely correlated with human health, and it has attracted a lot of attention.

[0003] The dramatic development of biotechnology research techniques, such as germ-free animal models, next-generation sequencing (NGS), and multi-omics analysis, has made it possible not only to analyze the composition and structure of intestinal microorganisms but also to study the relationship between microbiome function and disease, resulting in the publication of numerous research results. Microbiome therapeutics or permabiotic therapeutics (medical probiotics) have recently been attracting attention as potential alternative treatments for infectious diseases, immune disorders, metabolic disorders, and other conditions for which no effective treatments exist. If microbiome therapeutics or permabiotic therapeutics can be mass-produced and put into practical use, they are expected to be advantageously applicable to a variety of intractable diseases.

[0004] Because the human intestine is in an anaerobic state, most of the microorganisms that make up the microbiome are anaerobic. However, most of these microorganisms are strictly anaerobic, meaning that available carbon and nitrogen sources are very limited and they are extremely sensitive to oxygen. This makes it difficult to cultivate them at high concentrations and in large quantities for industrial purposes. Cultivating strictly anaerobic microorganisms is extremely difficult, and obtaining a high biomass yield is even more difficult.

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

[0006] Animal-derived ingredients are recognized as unsuitable for culturing anaerobic microorganisms for human food and pharmaceutical applications because they may contain contaminants of viral or bacterial origin, or may contain allergens, antigenic peptides, or other undesirable products. Despite considerable recent efforts, existing media for culturing anaerobic microorganisms are limited in that they are difficult to formulate, expensive, and unable to 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 overcomes the limitations and 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 produced stably over a long period of time with high yields when anaerobic microorganisms used as microbiome therapeutics or permabiotics are industrially mass-cultured, and that 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 a high final optical density. [Means for solving the problem]

[0009] One aspect of the present invention, which aims to solve the above-mentioned problems, relates to a medium supplement for high-yield culture of anaerobic microorganisms, which comprises 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 microorganisms are obligate anaerobic microorganisms in the human intestine, and may be, but are not necessarily 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 N-acetylhexosamine, an amino acid mixture of 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 of fructose, 2.5 g / L of lactose, 20 g / L of plant peptone, 10 g / L of yeast extract, 2.5 g / L of dipotassium hydrogen phosphate, 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.

[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] In order to solve the above-mentioned problems, still 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 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 for anaerobic microorganisms, the number of viable cells measured by the plate count method for the cultured anaerobic microorganisms is 10 10It is characterized by reaching a cell density of ≥ CFU / mL. [Effects of the Invention]

[0018] According to various embodiments of the present invention, anaerobic microorganisms used as microbiome therapeutics or permabiotics 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] The medium supplement and medium composition of the present invention make it possible to culture Akkermansia muciniphila, a difficult-to-cultivate bacterial species that is a promising permabiotic candidate but is extremely sensitive to oxygen and dies even in trace amounts of oxygen, making mass production impossible, at high concentrations suitable for pharmaceutical or food applications.

[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 explanation of the drawings]

[0021] [Figure 1] 1 is a schematic diagram showing the carbon metabolic process of Akkermansia muciniphila strains. [Figure 2] Schematic diagram showing the biosynthesis process of various amino acids from aspartic acid. [Figure 3] Photographs showing the cell morphology of anaerobic microorganisms classified by strain type cultured in a medium containing the medium supplement of the present invention. [Figure 4] 1 is a photograph showing the change in the color of the medium over the incubation time in an incubator in which an Akkermansia muciniphila strain was cultured in an example of the present invention. [Figure 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 the growth curve (A) of an Akkermansia muciniphila strain and the change in pH (B) according to the culture time in the medium of the present invention to which fructose has been added. [Figure 7] 1 is a graph showing the growth curve (A) of an Akkermansia muciniphila strain and the change in pH (B) according to the culture time in the medium of the present invention supplemented with maltose. [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 INVENTION

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

[0023] As used herein, 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 microorganism means the propagation of a microbial organism in a given culture medium under conditions conducive to its growth, thereby increasing it.

[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 microorganism" refers to a microorganism that is 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, recitation of numerical ranges by endpoints includes all numbers subsumed 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, and the medium supplement may contain 5 g / L of N-acetylhexosamine, 8 g / L of L-aspartic acid, 0.5 g / L of L-cysteine, and 0.0001 g to 0.005 g / L of cobalamin.

[0029] The medium supplement of the present invention is primarily used for culturing anaerobic microorganisms, which are 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. N-acetylhexosamines may include N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine (GalNAc), but preferably includes N-acetylglucosamine (GlcNAc). Figure 1 is a schematic diagram showing the carbon metabolism process of Akkermansia muciniphila. Referring to Figure 1, Akkermansia muciniphila contains beta-galactosidase, an enzyme that converts lactose to galactose and glucose, and alpha-glucosidase, an enzyme that converts maltose to glucose. Therefore, carbon sources such as lactose, maltose, and fructose can all undergo glycolysis and be used to form the high-energy molecule ATP. N-acetylglucosamine (Glc-NAc) supplied from the outside is used for cell wall synthesis (peptidoglycan biosynthesis) and energy metabolism. N-acetylglucosamine is metabolized to produce ammonia, which can 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 function of cells cultured in cell culture media. An external protein source is essential for sustaining good growth in high-density cultures of anaerobic microorganisms. 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. 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. Mucins that Akkermansia muciniphila can degrade are characterized by a repeating amino acid sequence rich in serine, threonine, proline, and cysteine. However, in the present invention, the addition of aspartic acid and cysteine ​​from these amino acids enables Akkermansia muciniphila to grow at high yields. Aspartic acid and cysteine ​​may exist in either the D- or L-form.

[0034] Figure 2 is a schematic diagram showing the biosynthesis process of various amino acids from aspartate. Referring to Figure 2, aspartate can be converted to homoserine, an intermediate in the biosynthesis of threonine and methionine. Various amino acids, including serine and proline, can be biosynthesized from aspartate. Furthermore, aspartate 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). Furthermore, the biosynthesis of lysine and threonine from aspartate is beneficial for the growth of some bacterial species, and aspartate intake can increase the diversity of intestinal microbiota.

[0035] The medium supplement composition of the present invention may contain aspartic acid and cysteine ​​in amounts ranging from about 4 to 8 g / L and about 0.5 to 1 g / L, respectively.

[0036] The medium supplement of the present invention contains cobalamin, i.e., vitamin B12. Cobalamin is used by cells as a cofactor. If the culture medium does not contain mucin, anaerobic microorganisms require a significant amount of cobalamin to grow to high densities. 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 contain cobalamin in an amount ranging from about 0.0001 to 0.005 g / L.

[0037] Vitamins such as biotin, choline chloride, folic acid, myo-inositol, niacinamide, pyridoxine HCl, D-pantothenic acid (hemiCa), riboflavin, and thiamine HCl are also used. However, the addition of cobalamin to the culture medium increases the relative abundance of anaerobic microorganisms, and the addition of other vitamins is not significantly different from the effect of adding cobalamin alone. Dielectric analysis of Akkermansia muciniphila revealed that most strains, including ATCC BAA-835 and EB-AMDK19, possessed genes related to the biosynthesis of B vitamins (B1, B2, B3, B5, B6, B7, and B9), but not those related to vitamin B12 biosynthesis. Furthermore, cobalamin in Akkermansia muciniphila may act as an important coenzyme for the synthesis of propionate from succinate.

[0038] Another aspect of the present invention relates to a media composition for the high-yield cultivation of anaerobic microorganisms comprising the media 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, a liquid medium is preferable when the purpose is industrial production by mass cultivation, but a medium containing plant peptone, yeast extract, and dipotassium hydrogen phosphate may also be used.

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

[0041] The medium composition of the present invention is suitable for culturing anaerobic microorganisms, specifically strict anaerobic microorganisms. Non-limiting examples of strict 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, at high yields 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 microbial culture of the present invention may contain a plant peptone. 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 cultivation 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 media of the present invention may contain phosphate-containing components, such as NaHPO 4、 The medium composition of the present invention further contains KHPO or KHPO. 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, and 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 optionally further contain maltose. 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, which is linked to galactose via a β(1→4)-glycoside bond, and N-acetylglucosamine and glucose can be interconverted in the metabolic pathway, so 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 predetermined volume of water to provide a desired concentration of a particular component in a liquid medium. Such powdered or concentrated medium may be dissolved in an appropriate amount of water, usually sterile water, before use.

[0049] In the present invention, media and media compositions include all final media containing components at concentrations suitable for culturing anaerobic microorganisms and powdered or concentrated media suitable 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 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 culture medium of the present invention may be achieved by replacing oxygen in the culture 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 culture medium may be 0.1 to 0.3 atmospheres, and preferably 0.2 atmospheres (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] As is well known to those skilled in the art, the culture conditions for microorganisms can affect the growth rate of the microorganisms. In the present invention, the culture step may be carried out at a pH of 6.6 to 7.0, a culture temperature of 35 to 39°C, an agitation speed of 40 to 50 rpm, a nitrogen saturation of 80 to 90%, a hydrogen saturation of 0 to 5%, and a carbon dioxide saturation of 5 to 20%.

[0054] In the present invention, the anaerobic microbial culture was measured at an optical density (OD ) 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 the plate count method is 10 10 It can be grown at high density to reach cell densities of CFU / mL or greater.

[0055] The culture temperature for high-concentration culture of anaerobic microorganisms is preferably 35 to 39°C, and particularly preferably 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 to 50 rpm, but is not limited to this. The culture period may be adjusted appropriately depending on the growth state of the anaerobic microorganisms, but is generally approximately 20 to 100 hours, and particularly approximately 24 to 48 hours.

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

[0057] In Akkermansia muciniphila, hydrogen ions are used for ATP synthesis in the metabolic pathway, and hydrogen (H2) is important for anaerobic respiration via Ni-dependent hydrogenase under anaerobic conditions. Therefore, for high-density cultivation of Akkermansia muciniphila EB-AMDK19 in the medium of the present invention, it is necessary to inject a hydrogen-containing gas mixture or a gas containing carbon dioxide, which 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 (biomass production) 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 value and after 48 hours (or 72 hours) of culture.

[0060] Example Example 1: Optimization of mucin-substitute component composition in culture medium The optimal combination of substrates and components to replace mucin was explored 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. The medium was then cultured at 37°C under anaerobic conditions (90% N2, 5% CO2, 5% H2) for 24-48 hours, and the optical density (OD 600The changes in the temperature were measured and the results are shown in Table 1 below.

[0061] [Table 1]

[0062] Although A. muciniphila strains barely grew in TSB medium, they were able to grow when N-acetylglucosamine (GlcNAc) and threonine were added as substitutes for mucin. However, when threonine was replaced with aspartate, the cultivability improved at the same concentration (4 g / L), and the growth rate increased up to a maximum of 8 g / L of aspartate.

[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 was at the level of 0.4, and the number of viable bacteria was 10 9 CFU / mL was measured, and 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, and it was found that there was a difference of more than 10 times.

[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 medium supplement of the present invention, based on plant peptone and yeast extract, was tested for anaerobic microbial culturability.

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

[0067] To investigate the influence of carbon sources on the cultivability of the anaerobic microorganism Akkermansia muciniphila, various carbon sources were added to the nitrogen-source basal medium as shown in Table 2. 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% N, 5% CO, 5% H) for 24-48 hours, after which the optical density (OD) was measured. 600 The changes in the temperature were 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 N-acetylglucosamine was added to lactose as a carbon source, or when N-acetylglucosamine and glucose were both added. The change in absorbance (△OD) was on average 0.5-0.6, 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 culturing ability was significantly improved in the present invention, with a 10-100-fold increase in the viable bacteria count. Furthermore, the addition of maltose, in which two glucose molecules are linked by a β(1→4)-glycoside, also improved the culturing ability compared to the addition of glucose.

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

[0072] Each prepared medium (see Table 4) 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 samples were cultured in 5% H2 for 24-48 hours and the optical density (OD 600 The changes in the temperature were 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 the B vitamin complex was added.

[0075] To find the optimal concentration of cobalamin, we also cultured the cells at different concentrations of cobalamin and measured the optical density (OD 600 The changes in the temperature were 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 culture medium was confirmed, and it was found that the cultivability gradually decreased at concentrations below 0.1 mg / L, and there was no significant difference in the cultivability between concentrations of 0.1 mg / L and 5 mg / L, so it is believed that the concentration of 0.1 mg / L is the most suitable.

[0078] Example 4: Optimization of medium components To explore the essential components and optimal combinations required for high-density cultivation of anaerobic microorganisms, Akkermansia muciniphila strains were cultured 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 examining the effect of each component on the culture of the medium of the present invention, it was confirmed that N-acetylglucosamine, a nitrogen-containing derivative of monosaccharides, is the most essential component for Akkermansia muciniphila culture, and that fructose and lactose, carbon sources that share part of the metabolic pathway with N-acetylglucosamine, are necessary components for high-density culture. The combination of soybean peptone, the main amino acid source, with aspartic acid and cyanocobalamin was confirmed to be an important component for improving culture.

[0081] Maltose can replace glucose and fructose, and the growth rate in the initial stage of cultivation is faster than that of fructose or 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 components of the medium of the present invention are necessary for high-density cultivation of Akkermansia muciniphila strains, and it was confirmed that the best cultivability was achieved when all components were combined.

[0082] Example 5: Cultivation of various Akkermansia muciniphila strains in the medium of the present invention The same procedure as in Example 1 was carried out, except that the medium described in Table 2 was used and the type of culture 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% N, 5% CO, 5% H) 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 type strain (ATCC BAA-835 T ) and different Akkermansia muciniphila strains were cultured. As a result, all Akkermansia muciniphila strains showed similar culture levels, and therefore it can be confirmed that it can be used as a high-concentration culture medium for all Akkermansia muciniphila strains.

[0085] Example 6: Comparison of the growth potential of various difficult-to-cultivate strictly anaerobic microorganisms The same procedure as in Example 1 was repeated except that the types of cultured strains were different as shown in Table 8 below. Do the same After inoculation with Faecalibacterium prausnitzii, Anaerostipes caccae, or Bifidobacterium longum strains at a ratio of 0.1% v / v, the mixture was cultured at 37°C under anaerobic conditions (90% N2, 5% CO2, 5% H2) for 24-48 hours, and the optical density (OD 600The changes in the β-amyloid activity (β-amyloid activity) of the culture medium of the present invention were measured, and the results are shown in Table 8 below. For comparison, the culture medium was also cultured in a control medium known to have excellent cultivability, and the cultivability 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 BL broth medium.

[0086] [Table 8]

[0087] As can be seen from the results in Table 8, the feasibility of culturing difficult-to-cultivate strictly anaerobic microorganisms such as Faecalibacterium prausnitzii and Anaerostipes caccae, and Bifidobacterium longum was examined. As a result, it was confirmed that the culture medium of the present invention has superior cultivability compared to the control medium, which is known to have excellent cultivability.

[0088] Example 7: Optimization of process conditions for large-scale cultivation - Comparative analysis of fertility under different cultivation conditions Using the anaerobic fermentation system shown in Figure 4, A. muciniphila was cultured at a 3L scale using the medium shown in Table 2 with different carbon sources under the conditions shown in Table 9. The growth curve and pH changes of the A. muciniphila strain were observed. Microscopic observation of changes in bacterial count and morphological changes were confirmed and shown in Figure 5. The viable bacterial count was measured using the plate count method and is shown in Table 10.

[0089] [Table 9]

[0090] [Table 10]

[0091] When Akkermansia muciniphila EB-AMDK19 strain was cultured in an anaerobic fermentation system using the medium of the present invention, it was found that the exponential growth phase occurred 5 to 25 hours after inoculation, and a rapid change in pH was also observed 9 to 18.5 hours after inoculation. The optical density (OD) was measured based on the 24-hour culture. 600 ) is found to be at the 0.798 level, and the number of live bacteria at this time is 5.5x10 10 CFU / mL (see Figure 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 a rapid change in pH was also confirmed to occur 7 to 14 hours after inoculation. Compared with the results of culturing using fructose, the medium of the present invention containing maltose showed a characteristic that the lag phase was shortened by 1.5 hours, and the absorbance was slightly lower at 0.632 after 24 hours of culturing, but the viable cell count at this time was 5.1 x 10 10 There was no significant difference from CFU / mL (see Figure 7 and Table 9). In the fructose-containing medium of the present invention, cultivation was carried out using a more economical and industrially suitable hydrogen-free gas (high-purity nitrogen gas (100% N) or a mixed gas composed of 80% N and 20% CO) instead of a mixed gas (90% N, 5% CO, 5% H). As a result, it was found that the culturing ability was significantly inferior to the conventional culture results when using high-purity nitrogen gas, but there was no significant difference in the culturing ability when using a gas composed of 80% N and 20% CO (see FIG. 8 and Table 10).

[0093] Furthermore, as can be seen from Table 10, all of the components in the medium of the present invention are necessary for high-density cultivation of Akkermansia muciniphila strains, and it was confirmed that injection of a mixed gas containing hydrogen and carbon dioxide is also necessary.

Claims

1. A medium composition for culturing an anaerobic microorganism, comprising plant peptone, yeast extract, and dipotassium hydrogen phosphate, the medium containing lactose and at least one of fructose and maltose as a carbon source, and containing N-acetylhexosamine, an amino acid mixture of L-aspartic acid and L-cysteine, and cobalamin as supplements; the N-acetylhexosamine is N-acetylglucosamine (GlcNAc), The anaerobic microorganism is Akkermansia muciniphila, Faecalibacterium prausnitzii, or Anaerostipes cacae. A medium composition for culturing anaerobic microorganisms.

2. 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, broad bean peptone, lupin peptone, and potato peptone.

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

5. 5. The method for culturing anaerobic microorganisms according to claim 4, wherein the culturing step is carried out at a pH of 6.6 to 7.0, a culture temperature of 35 to 39°C, an agitation speed of 40 to 50 rpm, a nitrogen saturation of 80 to 90%, a carbon dioxide saturation of 5 to 20%, and a hydrogen saturation of 0 to 5%.

6. 6. The method for culturing anaerobic microorganisms according to claim 5, wherein the cultured anaerobic microorganisms reach a cell density of 10<10> CFU / mL or more as a viable cell count measured by plate counting.

7. The method for culturing anaerobic microorganisms according to claim 4, characterized in that the anaerobic microorganisms are Akkermansia muciniphila, Faecalibacterium prausnitzii, or Anaerostipes cacae.

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