Culture medium composition for Faecalibacterium prausnitzii strain

Abstract

The present invention relates to a medium composition for culturing Faecalibacterium prausnitzii, which comprises vegetable peptone, yeast extract, a phosphate compound, a carbonate compound, cyanocobalamin (B12), L-cysteine, ammonium acetate, and maltose, and a method for mass-cultivating Faecalibacterium prausnitzii using the same. The medium composition and the method for cultivating Faecalibacterium prausnitzii according to the present invention enable high-concentration cultivation and mass production of Faecalibacterium prausnitzii phylogroup II strains. Furthermore, since the culture medium composition and the method for cultivating Faecalibacterium prausnitzii according to the present invention enable high-concentration cultivation and mass production even in high-concentration anaerobic nitrogen gases while eliminating animal components, the culture method is more economical and suitable for industrial application.

Description

[Technical field]

[0001] The present invention relates to a culture medium composition for a Faecalibacterium prausnitzii strain. [Background technology]

[0002] Faecalibacterium prausnitzii is the most common butyrate-producing bacterium in the gastrointestinal tract, and it is very important to maintain the number of Faecalibacterium prausnitzii in the intestine at a certain level. In fact, it has been observed that the number of Faecalibacterium prausnitzii decreases rapidly in patients with Crohn's disease and ulcerative colitis, and it has been reported that this is also involved in the formation of O-glycans in the mucus layer and has an important effect on the physiological function of the intestine. Therefore, if we can mass-produce and ingest such Faecalibacterium prausnitzii, we should be able to ensure butyrate in the intestine.

[0003] Recently, in a study focusing on the diversity of the genus Faecalibacterium, Lopez-Siles et al. analyzed the phylogenetic relationships of Faecalibacterium prausnitzii isolates based on 16S rRNA gene sequences and identified two phylogroups (phylogroups I and II) within the species Faecalibacterium prausnitzii. It has been reported that even within the same species, the utilization ability of carbon sources (glucose, cellobiose, maltose, galacturonic acid, galactose, apple pectin, insulin, glucuronic acid, N-acetylgucosamine, etc.) differs between phylogroups (Lopez-Siles et al. Appl Environ Microbiol., 2012; 78: 420-428).

[0004] All species of Faecalibacterium prausnitzii were reported to possess beta-galactosidase, alpha-glucosidase, and phosphatase activities, but not urease, arginine dihydrolase, beta-glucosidase, and alpha-fucosidase activities. They were able to grow in YCFA medium supplemented with fructose, fructo-oligosaccharides, and glucose as substrates, but were unable to grow on arabinose, melibiose, raffinose, rhamnose, ribose, and xylose (Duncan et al., 2003). al., 2002). Because they possess the enzymes beta-galactosidase, which converts lactose to galactose and glucose, and alpha-glucosidase, which converts maltose to glucose, any carbon source such as lactose or maltose can be used to form the high-energy molecule ATP via glycolysis.

[0005] Amino acids are important for maintaining the metabolic function of cells cultured in cell culture media, and an external protein source is essential to sustain good growth during high-density culture. The amino acid source may be any known amino acid source, including but not limited to animal, plant, or microbial amino acid sources, and ideally entirely plant or microbial. The amino acid source may also be, for example, a protein hydrolysate, a plant-derived protein hydrolysate. Protein hydrolysates are produced from protein sources using hydrolysis and typically consist of a mixture of peptides, amino acids, carbohydrates, and lipids, as well as a number of unidentified components with vague biological activity. They are often produced by enzymatic, alkaline, or acidic degradation of raw materials provided from a variety of sources, including but not limited to plant sources (soybean, wheat, pea, chickpea, or cotton). The amino acid source may also be an amino acid composition that includes individual amino acids or combinations of individual amino acids.

[0006] Currently, the cultivation technology for the Faecalis bacterium prausnitzii species is 10 9 The current level remains at the CFU / mL level, which is a hurdle to developing a mass production process. 11 High-concentration cultivation at the CFU / mL level is expected to become a very important technology in the industrialization stage.

[0007] Strains belonging to Faecalibacterium prausnitzii phyllogroup II show a very different cell morphology from strains belonging to phyllogroup I. In particular, strains belonging to Faecalibacterium prausnitzii phyllogroup II have been confirmed to show long-chain rod-shaped cells when cultured in a medium containing amino sugar such as N-acetyl-D-glucosamine (GlcNAc). As a result, the cell number increase is limited in the limited space, so it is necessary to culture the cells in a medium containing 10 11 It is difficult to culture at high concentrations at CFU / mL levels. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Lopez-Siles et al.Appl Environ Microbiol.,2012;78:420-428 Summary of the Invention [Problem to be solved by the invention]

[0009] One embodiment provides a medium composition for culturing Faecalibacterium prausnitzii, comprising vegetable-peptone, yeast extract, a phosphate compound, a carbonate compound, cyanocobalamin, L-cysteine, ammonium acetate, and maltose.

[0010] Another aspect provides a method for culturing Faecalibacterium prausnitzii, comprising the steps of inoculating the medium composition with Faecalibacterium prausnitzii and culturing the inoculated strain under conditions of 35 to 38°C, pH 6.5 to 7, and a gas flow rate of 0.1 to 0.3 L / h. [Means for solving the problem]

[0011] One embodiment provides a medium composition for culturing Faecalibacterium prausnitzii, comprising vegetable-peptone, yeast extract, a phosphate compound, a carbonate compound, cyanocobalamin, L-cysteine, ammonium acetate, and maltose.

[0012] According to one embodiment, the carbonate compound may be in a concentration of 2-6 g / L.

[0013] According to one embodiment, the maltose may be at a concentration of 2.5 to 30 g / L.

[0014] According to one embodiment, the ammonium acetate may be at a concentration of 2 to 16 g / L.

[0015] In one embodiment, the Faecalibacterium prausnitzii may be Faecalibacterium prausnitzii Phylogroup II.

[0016] Another aspect provides a method for culturing Faecalibacterium prausnitzii, comprising: a) the steps of inoculating Faecalibacterium prausnitzii into the medium composition described in claim 1; and b) culturing the inoculated strain under conditions of 35 to 38°C, pH 6.5 to 7, and a gas flow rate of 0.1 to 0.3 L / h. Effect of the Invention

[0017] When the medium composition and the method for culturing Faecalibacterium prausnitzii according to the present invention are used, it is possible to culture and mass-produce strains of the Faecalibacterium prausnitzii phyllogroup at high concentrations. In addition, since the strains can be cultured at high concentrations even in high concentrations of anaerobic nitrogen gas during mass cultivation while eliminating animal components, it is possible to provide a more economical and industrially suitable culture method. [Brief description of the drawings]

[0018] [Figure 1] This is a photograph of Faecalibacterium prausnitzii cultured with glucose (GlU) and N-acetylglucosamine (GlcNAc) or a combination of alternative components in order to find an alternative to N-acetylglucosamine (GlcNAc) (GLU: glucose, GlcNAc: N-acetylglucosamine, NaCAS: sodium caseinate). [Diagram 2]This is a photograph of Faecalibacterium prausnitzii cultured with glucose (GlU) and N-acetylglucosamine (GlcNAc) or a combination of alternative components to find an alternative to N-acetylglucosamine (GlcNAc) (GLU: glucose, FOS: fructooligosaccharides, PEC: pectin, CEL: cellulose, CHI: chitin, TRE: trehalose). [Diagram 3] This is a photograph of the strain viewed under a microscope after culturing Faecalibacterium prausnitzii in a medium containing a combination of sodium caseinate and a carbon source (NaCAS: sodium caseinate, FRU: fructose, GAL: galactose, LAC: lactose, CBS: cellobiose, MAL: maltose, SPV: sodium pyruvate). [Figure 4] This is a diagram showing the regulation process of maltose uptake in the literature, An integrated transport mechanism of the maltose ABC importer (Machtel et al., 2019, Res. Microbiol., 190:321-337.). A shows the process when PTS sugar is present, in which PTS sugar using the transporter restricts maltose uptake due to the activity of PTS permease, and B shows the process when PTS sugar is not present, in which PTS permease is not activated and maltose uptake is activated. [Diagram 5] The data from the genome analysis of Faecalibacterium prausnitzii strains confirmed that phylogroup II (A2-165, EB-FPDK11, EB-FPYYK1) has a higher number of copies of ABC transporter-related genes than phylogroup I (ATCC27768, EB-FPDK9) and the unclassified group (EB-FPDK3). [Figure 6] This data shows the degree of similarity between the genomes of strains based on ANI (average nucleotide identity). [Figure 7] The data was obtained by culturing Faecalibacterium prausnitzii in medium supplemented with sodium caseinate and cellobiose (CBS), maltose (MAL), maltodextrin (MDX), or pullulan (PUL), and then examining the strain under a microscope. [Figure 8] This data was obtained by culturing Faecalibacterium prausnitzii in a medium containing sodium caseinate, cellobiose (CBS), maltose (MAL), maltodextrin (MDX), and pullulan (PUL), and then examining the strain under a microscope. [Figure 9] This data was obtained by comparing the appearance of Faecalibacterium prausnitzii cultured when sodium acetate (SoAc) or ammonium acetate (AmAc) was added to the medium under a microscope. [Figure 10] These are microscopic data of Faecalibacterium prausnitzii cultured in a medium lacking one of maltose (MAL), ammonium acetate (AmAc), cyanocobalamin (CYA), and sodium caseinate (NaCAS). [Figure 11] The data confirm that Faecalibacterium prausnitzii can be cultivated at high concentrations in the medium of the present invention, and are obtained by comparing the viable cell counts when cultured in YBHI w / CB, MT medium (Unoptimized), and SPYE w / GSGC medium (a medium containing soy-peptone, yeast extract, glucose, dipotassium phosphate, GlcNAc, sodium acetate, and cyanocobalamin, and containing 0.05% w / v L-cysteine; hereinafter referred to as "SPYE w / GSGC medium") disclosed in Korean Patent No. 10-2245415. [Figure 12]The data were obtained by culturing Faecalibacterium prausnitzii phylogroup II strains (A2-165, EB-FPYYK1), Faecalibacterium prausnitzii phylogroup I strains (EB-FPDK9), and Faecalibacterium prausnitzii unclassified group strains (EB-FPDK3) in SPYE w / GSGC and SPYE w / MACN medium (medium containing soy-peptone, yeast extract, dipotassium phosphate, maltose, ammonium acetate, cyanocobalamin, sodium caseinate, and 0.05% w / v L-cysteine; hereafter referred to as "SPYE w / MACN medium") and observing them under a microscope. [Figure 13] FIG. 1 is a diagram showing a predicted process by which the medium composition of the present invention is metabolized by Faecalibacterium prausnitzii. [Figure 14] 1A is data showing OD values ​​as a function of culture time in SPYE w / GSGC and SPYE w / MACN media, and B is data showing pH values ​​as a function of culture time in SPYE w / GSGC and SPYE w / MACN media. [Figure 15] This shows data obtained by microscopic observation of the bacterial strains cultured in large-scale culture in SPYE w / GSGC and SPYE w / MACN medium compositions. [Figure 16] The data were obtained by culturing Faecalibacterium prausnitzii phylogroup II strains (EB-FPDK11, EB-FPYYK1, A2-165) under conditions containing phosphate and carbonate, and then observing them under a microscope. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] One embodiment provides a medium composition for culturing Faecalibacterium prausnitzii, comprising vegetable peptone, yeast extract, a phosphate compound, a carbonate compound, cyanocobalamin (B12), L-cysteine, ammonium acetate, and maltose.

[0020] The soy peptone and yeast extract are plant- or microorganism-derived components that replace animal-derived components, which may contain viral or bacterial contaminants, allergens, or antigen peptides, and are therefore unsuitable for use in biopharmaceuticals and health functional foods.

[0021] The plant peptone may be peptone extracted from a plant, such as, but not limited to, soy peptone, wheat peptone, cotton peptone, pea peptone, broadbean peptone, lupin peptone, and potato peptone. The plant peptone may be included in the medium composition in an amount of 10 g / L to 20 g / L or 15 g / L to 20 g / L.

[0022] The yeast extract can serve as a protein source to further increase the growth of anaerobic microorganisms on non-animal media. The yeast extract can be a yeast autolysate, an ultrafiltered yeast extract, or a synthetic yeast extract. The concentration of the yeast extract can be 5 g / L to 10 g / L, for example, 10 g / L.

[0023] The phosphate compound may be disodium phosphate (Na2HPO4), dipotassium phosphate (K2HPO4) or potassium phosphate (KH2PO4). The phosphate compound is a substance added to a 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, 6.0 to 7.0, or 5.8 to 7.8, but is not limited thereto.

[0024] According to one embodiment, the carbonate compound may have a concentration of 2 to 6 g / L, for example, 2 to 4 g / L, 2 to 5 g / L, 3 to 4 g / L, 3 to 5 g / L, 3 to 6 g / L, 4 to 5 g / L, or 4 to 6 g / L.

[0025] The carbonate compound is a component that acts as a buffer together with the phosphate compound and produces CO2, which increases the economy during mass cultivation.

[0026] According to one embodiment, the maltose may have a concentration of 2.5 to 30 g / L, for example, 2.5 to 25 g / L, 2.5 to 20 g / L, 2.5 to 15 g / L, and 2.5 to 12.5 g / L.

[0027] According to one specific example, the ammonium acetate may have a concentration of 2 to 16 g / L, such as 2 to 16 g / L, 2 to 12 g / L, 2 to 10 g / L, 4 to 16 g / L, 4 to 12 g / L, and 4 to 10 g / L.

[0028] As is well known by ordinary skilled artisans, the culture conditions of microorganisms can affect the growth rate of the microorganisms. In the present invention, the culture step can be carried out at pH 6.6-7.0, culture temperature 36°C-39°C, stirring speed 40 rpm-50 rpm, nitrogen saturation 80%-100%, hydrogen saturation 0%-5%, and carbon dioxide saturation 0%-20%.

[0029] The Faecalibacterium prausnitzii includes all of the phylogroups I, IIa, and IIb of Faecalibacterium prausnitzii, and may be, for example, but not limited to, Faecalibacterium prausnitzii EB-FPDK9 of phylogroup I, Faecalibacterium prausnitzii A2-165 and Faecalibacterium prausnitzii EB-FPYYK1 of phylogroup IIa, Faecalibacterium prausnitzii EB-FPDK11 of phylogroup IIb, and Faecalibacterium prausnitzii EB-FPDK3 of the unclassified group.

[0030] According to one embodiment, the Faecalibacterium prausnitzii may be Faecalibacterium prausnitzii Phylogroup II, for example, Faecalibacterium prausnitzii EB-FPDK11.

[0031] Another aspect provides a method for culturing Faecalibacterium prausnitzii, comprising: a) the steps of inoculating Faecalibacterium prausnitzii into the medium composition described in claim 1; and b) culturing the inoculated strain under conditions of 35 to 38°C, pH 6.5 to 7, and a gas flow rate of 0.1 to 0.3 L / h.

[0032] The description of Faecalibacterium prausnitzii is the same as that described above. EXAMPLES

[0033] Hereinafter, one or more embodiments will be described in more detail with reference to examples. However, these examples are merely for illustrative purposes and are not intended to limit the scope of the present invention.

[0034] Example 1. Confirmation of cell morphological changes caused by medium supplements In relation to the cultivation of Faecalibacterium prausnitzii, we observed morphological changes in the cells of Faecalibacterium prausnitzii phylogroup II strains in culture media supplemented with various substrates known from existing literature (Duncan et al., 2002; Lopez-Siles et al., 2012, Lopez-Siles et al., 2017).

[0035] As the strain of Faecalibacterium prausnitzii phylogroup II, Faecalibacterium prausnitzii EB-FPDK11, which is deposited under Korean Patent Registration No. 10-2169794 and assigned the accession number KCCM12621P, was used.

[0036] The medium composition of Korean Patent No. 10-2245415 contains 20 g / L soy peptone, 10 g / L yeast extract, 2.5 g / L glucose, 2.5 g / L dipotassium phosphate, 2.5 g / L N-acetyl-D-glucosamin (GlcNAc), 2 g / L sodium acetate, and cyanocobalamin (B 12 The culture properties of the medium containing 0.1 mg / L of SPYE and 0.05% w / v L-cysteine ​​(hereinafter referred to as "SPYE w / GSGC") were compared and analyzed.

[0037] For this purpose, the isolated strain Faecalibacterium prausnitzii EB-FPDK11 was inoculated into each prepared medium at a ratio of 0.1% v / v, and then cultured at 37°C under anaerobic conditions (90% N2, 5% CO2, 5% H2) for 24-48 hours, and the change in turbidity (OD600nm) was measured. The following SPYE medium contains 20g / L soy-peptone, 10g / L yeast extract, 2.5g / L dipotassium phosphate, 2g / L sodium acetate, cyanocobalamin, and 10g / L ethanol. 12 The medium contained 0.1 mg / L of glucose (Glu), N-acetylglucosamine (GlcNAc), sodium caseinate (NaCAS), fructooligosaccharides (FOS), pectin (PEC), cellulose (CEL), chitin (CHI), and trehalose (TRE) as shown in Table 1 below, and the turbidity and cell shape of each medium were confirmed.

[0038] [Table 1]

[0039] As can be seen from Table 1 and Figures 1 and 2, the Faecalis bacterium prausnitzii EB-FPDK11 strain was confirmed to show relatively short and straight cell morphology in the medium without GlcNAc, and the absorbance was slightly lower, but the cell density (cell number) did not show a significant difference. In addition, as can be seen from the results of the medium with sodium caseinate (NaCAS), it was confirmed that sodium caseinate can compensate to some extent for the decrease in cultivability when N-acetylglucosamine (GlcNAc) is not added.

[0040] Example 2. Search for optimal carbon source combination When sodium caseinate (NaCAS) was used in place of N-acetylglucosamine (GlcNAc) in SPYE medium, the optimal carbon source was identified by combining glucose (GLU), fructose (FRU), galactose (GAL), lactose (LAC), cellobiose (CBS), maltose (MAL), and sodium pyruvate (SPV) as shown in Table 2 below.

[0041] [Table 2]

[0042] As shown in Table 2 and Figure 3, when cellobiose and maltose were added instead of glucose in a medium not containing N-acetylglucosamine, the cells were observed to have a very short rod-like shape, and especially when maltose was used instead, the cells showed a very small morphology, confirming the possibility of high-density culture that allows the cell number to increase in a limited space. Similarly, it was revealed that the absorbance decreased due to the decrease in cell morphology and size, but the cell density (cell number) did not show a significant difference.

[0043] In the case of maltose, ABC transporter is used, but in the presence of sugars that use PTS transporter (PTS sugar: Glucose, Fructose, GlcNAc, etc.), absorption of maltose is limited by the activity of PTS permease, as can be seen in Figure 4, an excerpt from An integrated transport mechanism of the maltose ABC importer (Machtel et al., 2019, Res. Microbiol., 190:321-337.).

[0044] In addition, as shown in Figures 5 and 6, genome analysis of Faecalibacterium prausnitzii strains confirmed that phyllogroup II strains (A2-165, EB-FPDK11, EB-FPYYK1) had relatively higher copy numbers of ABC transporter-related genes than phyllogroup I strains (ATCC27768, EB-FPDK9) and the unclassified group EB-FPDK3, suggesting that substrates such as maltose may be useful for cultivation.

[0045] Example 3. Confirmation of whether culturing can be improved by culture concentration In the above examples, maltose, which showed high potential for high-density cultivation, and other polymers such as maltodextrin (MDX), pullulan (PUL), and cellobiose (CBS) were added to the SPYE medium at different concentrations to confirm whether they could improve the culturing ability.

[0046] [Table 3]

[0047] As shown in Table 3, Figures 7 and 8, when maltose was added instead of glucose to a medium without N-acetylglucosamine, the cells were observed to have a very short rod-like shape, and the absorbance increased significantly in a concentration-dependent manner, reaching a level equivalent to that of the existing medium (SPYE w / GSGC) at maltose concentrations of 12.5 g / L to 25 g / L. In particular, microscopic observation revealed that the cell density (cell number) had increased significantly.

[0048] When cellobiose or maltodextrin was added, the cells showed a slightly longer morphology than when only maltose was added, and when pullulan was added, the cells showed a very short morphology, similar to when maltose was added, but the absorbance and cell density were very low, and it was confirmed that the medium was not suitable as a culture medium for Faecalibacterium prausnitzii.

[0049] Example 4. Improvement of culturability by adding ammonium acetate Acetate salts of sodium acetate (SoAc) added to existing culture media are used as a substrate for producing butyrate and are known as growth factors. In addition, N-acetylglucosamine is used in cell wall synthesis (peptidoglycan biosynthesis) and energy metabolism, and produces ammonia during the metabolic process. Ammonia acts as a pH buffer to neutralize the cytoplasm and functions as a nitrogen source. Therefore, it was found that maltose medium without N-acetylglucosamine is very sensitive to pH changes, and the pH after cultivation was usually confirmed to be 6.1 to 6.3. It was observed that when the pH of the maltose medium drops below 6, the morphology of the cells changes and the culturing ability is poor. Therefore, ammonium acetate (AmAc), a growth factor that can act as a pH buffer and supply a nitrogen source and acetate, was added instead of sodium acetate to confirm whether the culturing ability could be improved. To confirm whether the culturing ability was improved, the basal medium was prepared based on the results of the previous embodiment, containing 20 g / L soy peptone, 10 g / L yeast extract, 2.5 g / L dipotassium phosphate, 12.5 g / L maltose, 2.5 g / L sodium caseinate, cyanocobalamin, and 1.0 g / L glycerol. 12 The medium used herein was a medium containing 0.1 mg / L of SPYE and pretreated with 0.05% w / v L-cysteine ​​(hereinafter, "SPYE w / MCN").

[0050] [Table 4]

[0051] As can be seen from Table 4 and Figure 9, in the medium where ammonium acetate was added instead of sodium acetate, it was found that the culturability increased in a concentration-dependent manner, and in particular, at a concentration of 6g / L, the absorbance reached a maximum, the cells were observed to have a very short rod-like shape, and it was confirmed that the cell density (cell number) had increased significantly. In the medium where ammonium acetate was added at a concentration of 10g / L or more, a culturability inhibition phenomenon rather occurred, and it was confirmed that proliferation was not possible at a concentration of 20g / L.

[0052] Example 5. Confirmation of the importance of each component of the medium supplement The effect of each component of the proposed combination of substrates (maltose, ammonium acetate, cyanocobalamin, and sodium caseinate) on the culture was evaluated.

[0053] [Table 5]

[0054] As can be seen from Table 5 and FIG. 10, when any one of the four components was removed, the culturing ability was significantly decreased, and it was confirmed by microscopic observation that the cell density (cell number) was significantly decreased.

[0055] Example 6. Confirmation of improved culturability of phyllogroup II strains The cultureability of different strains of Faecalibacterium prausnitzii phylogroup II was examined using the optimized medium derived in Example 5. To compare the effects, the EB-FPDK9 strain of phylogroup I and the EB-FPDK3 strain, which does not belong to phylogroups I and II, were used.

[0056] [Table 6]

[0057] As shown in Table 6 and FIG. 11, in the present invention, the strain of Faecalis prausnitzii phyllogroup II was cultured for 10 min in a SPYE w / MACN medium (a combination of soy peptone at a concentration of 20 g / L, yeast extract at a concentration of 10 g / L, dipotassium phosphate at a concentration of 2.5 g / L, maltose at a concentration of 12.5 g / L, ammonium acetate at a concentration of 6 g / L, cyanocobalamin at a concentration of 0.1 mg / L, and sodium caseinate at a concentration of 2.5 g / L). 10 ~10 11 It was confirmed that high-concentration cultivation was possible in CFUs / mL.

[0058] In addition, as shown in Figure 12, microscopic observation confirmed that A2-165 and EB-FPYYK1, which belong to Faecalibacterium prausnitzii phyllogroup II, became shorter and more dense in SPYE w / MACN medium compared to SPYE / GSGC medium. In contrast, EB-FPDK9, which belongs to Faecalibacterium prausnitzii phyllogroup I, and EB-FPDK3, which belongs to the unclassified group, did not show a significant difference in cultivability compared to SPYE / GSGC medium, or rather, the strains grew longer and could not be cultured at high density.

[0059] Through the present invention, it is estimated that the medium components consisting of a combination of soy peptone, yeast extract, maltose, ammonium acetate, and cyanocobalamin sodium caseinate are metabolized by Faecalibacterium prausnitzii through the metabolic pathway shown in Figure 13, thereby improving cost-effectiveness by replacing expensive N-acetylglucosamine.

[0060] Example 7. Confirmation of whether mass culture is possible Using a customized anaerobic fermenter system capable of confirming and maintaining anaerobic conditions for the cultivation of EOS (extremely oxygen sensitive) bacteria such as Faecalis bacterium prausnitzii, large-scale cultivation was achieved at a fermentation scale of 3 liters with the established medium composition (Table 6). For this purpose, cultivation was carried out under the composition in Table 7 and conditions in Table 8 below, and the growth curve and pH change were confirmed, and morphological changes were observed under a microscope and the viable cell count was confirmed using a plate count method.

[0061] [Table 7]

[0062] [Table 8]

[0063] As a result, as shown in FIG. 14, the Faecalis bacterium prausnitzii EB-FPDK11 strain reached maximum absorbance after 22.5 hours of mass culture using a fermenter, and the absorbance (OD600) at this time was 1.23, which was the same level as that of test tube culture. This was more than twice as high as that of the medium (SPYE w / GSGC) of Korean Patent No. 10-2245415, and the viable cell count was 2.0×10 11 CFUs / mL and 2.04 × 10 9 It was revealed that the CFUs / mL increased 100-fold. Similarly, as a result of microscopic observation, as shown in Figure 15, the SPYE w / MACN cultured cells showed a very short and small rod-like cell morphology compared to the SPYE w / GSGC medium, and it was confirmed that the cell density (cell number) was also significantly improved.

[0064] Example 8. Search for culture conditions that consider the efficiency and economy of mass production Because microbial ATP synthesis and butyrate production pathways require hydrogen ions, carbon dioxide, which dissolves in water to produce carbonic acid and hydrogen ions, is known to be an essential component of a suitable gas mixture for the growth of anaerobic microorganisms (Killam et al. J. Clin. Microbiol., 2003; 41: 2201-2202, Reilly et al., J. Med. Microbiol., 1980; 13: 573-579, Stalons et al., Appl. Microbiol. 1974; 27: 1098-1104).

[0065] The commonly known composition of the culture gas for Faecalibacterium prausnitzii is 90%N2+5%CO2+5%H2, 80%N2+10%CO2+10%H2, 81%N2+11%CO2+8%H2, and 97%CO2+3%H2, containing 5% to 97% CO2. However, there is a problem that it is not easy to add CO2 during large-scale culture, which makes it less economical. If culture is performed only in 100% N2 to overcome this economical problem, as can be seen from Korean Patent No. 10-2245415, if Faecalibacterium prausnitzii is cultured in anaerobic gas composed of only 100% N2, the lag phase is significantly delayed.

[0066] In order to overcome the economical disadvantages of mass cultivation systems by culturing in high-purity nitrogen (100% N2) anaerobic gas, and to confirm whether high-concentration cultivation is possible without using sodium caseinate, an animal-derived ingredient, phyllogroup II strains were cultivated and the differences in culturability in the presence or absence of carbonate compounds and sodium caseinate were compared.

[0067] Based on the results of the above example, the basal medium was prepared by adding 20 g / L soy peptone, 10 g / L yeast extract, 12.5 g / L maltose, 6 g / L ammonium acetate, and cyanocobalamin (B). 12The medium used was pretreated with 0.05% w / v L-cysteine ​​(hereinafter, "SPYE w / MAC") and contained 0.1 mg / L of L-cysteine. As shown in Table 9 below, the buffering action of phosphate compounds (K2HPO4, KH2PO4) and CO2 + H2 ⇔ H2CO3 ⇔ HCO3 - +H + From the reaction equation, a carbonate compound (KHCO3) was derived that can act as a buffer and generate hydrogen ions and CO2, and culturing was confirmed in a medium that combined the carbonate compound (KHCO3). In the case of carbonate compounds, CO2 is generated by thermal decomposition, so they were added separately by filtration sterilization rather than high-pressure steam sterilization. In addition, as can be seen from Table 10 below, it was confirmed that potassium bicarbonate, which has a higher thermal decomposition temperature and solubility, is more suitable for large-scale fermentation than potassium bicarbonate (KHCO3) and sodium bicarbonate (NaHCO3), which are typical carbonate compounds.

[0068] [Table 9]

[0069] [Table 10]

[0070] [Table 11]

[0071] As can be seen from Table 11 and Figure 16, under 100% N2 conditions, the presence or absence of potassium bicarbonate significantly affected culturing. The EB-FPDK11 strain exhibited maximum absorbance at a concentration of 2 g / L, and the cells were observed to have a very short rod-like shape. In addition, when calculating the viable cell count with or without sodium caseinate, the number of viable cells was 3.2 x 10 11 CFUs / mL and 5.3 × 10 11Under 100% N2 conditions, the increase in absorbance was greatest for the EB-FPYYK1 and A2-165 strains at a potassium bicarbonate concentration of 0.4 g / L rather than 0.2 g / L, and the viable cell count of the EB-FPYYK1 strain was 3.0 × 10 10 CFUs / mL, viable cell count of A2-165 strain was 2.7×10 11 However, when combined with sodium caseinate, the culturability was significantly reduced, showing a slightly different pattern from that of the EB-FPDK11 strain.

[0072] In conclusion, we confirmed that the combination of phosphate and carbonate compounds enables culture under CO2-free gas conditions, and that the culture medium composition does not contain any animal-derived components. 11 It was confirmed that high-concentration cultivation at the CFUs / mL level was possible.

Claims

1. Vegetable peptone, yeast extract, phosphate compounds, carbonate compounds, cyanocobalamin (B12), L-cysteine, ammonium acetate, and maltose. A medium composition for culturing Faecalibacterium prausnitzii, comprising: the plant peptone is at a concentration of 10 to 20 g / L; the yeast extract is at a concentration of 5-10 g / L; the carbonate compound is at a concentration of 2 to 6 g / L; the ammonium acetate is at a concentration of 2 to 16 g / L; and The maltose has a concentration of 7.5 to 37.5 g / L; A medium composition for culturing Faecalibacterium prausnitzii.

2. The Faecalibacterium prausnitzii is Faecalibacterium prausnitzii phylogroup II; The medium composition for culturing Faecalibacterium prausnitzii according to claim 1.

3. A method for culturing Faecalibacterium prausnitzii, comprising the steps of: a) inoculating the medium composition of claim 1 with Faecali bacterium prausnitzii; b) culturing the inoculated strain under conditions of 35-38° C., pH 6.5-7, and gas flow rate of 0.1-0.3 L / h; A method for culturing Faecalibacterium prausnitzii.

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