KR20230086143A - Culture media for producing high yield of Akkermansia sp., culturing method and freeze drying method using it - Google Patents

Abstract

The present invention relates to a medium composition for culturing microorganisms of the genus Akkermansia, which includes N-Acetylhexosamine, L-Threonine, and a yeast extract, and to a medium composition for culturing microorganisms of the genus Akkermansia, which includes disaccharides as a sole carbon source and animal protein hydrolyzate as a nitrogen source and a method for culturing microorganisms of the genus Akkermansia using the same. The present invention enables the culturing of microorganisms of the genus Akkermansia such as Akkermansia muciniphila, an unculturable anaerobic microorganism, with high yield and enables the production of freeze-dried bacterial powder with improved survival rate and stability for the cultured microorganisms of the genus Akkermansia.

Description

Culture media for producing high yield of Akkermansia sp., culturing method and freeze drying method using it}

The present invention is to provide a medium composition for culturing egg-cultivated anaerobic microorganisms, a culture method using the same, and a method for producing a freeze-dried powder prepared using the same.

The human microbiome is a term that refers to a community of microorganisms that live symbiotically in the human body and the genome of such a community of microorganisms, and it has been revealed that the microbiome is closely related to human health.

Since the microorganisms constituting the human microbiome are anaerobic in the human intestine, most of them are anaerobic microorganisms, and among them, Akkermansia genus microorganisms, especially Akkermansia muciniphila , are Korean Patent No. 1809172 It has been found to be effective in the treatment of metabolic diseases in Ho, and is also known to be effective in the treatment of degenerative brain diseases in Korean Patent No. 1799829.

The Akkermansia genus microorganisms are obligate anaerobic microorganisms that are extremely sensitive to oxygen, and can be cultured by adding hog gastric mucin extracted from the stomach of pigs as carbon and nitrogen sources to a general medium (Derrien et al., 2004) , It is known that mucin can be added and cultured on Columbia broth (CB) and brain heart extract (BHI) broth, but the available carbon and nitrogen sources are very limited, so the production yield is high to an industrially useful extent, and economically mass-produced It is an extremely difficult situation.

U.S. Patent No. 10,988,809 proposes a method for culturing Ackermansia municipilla using a medium containing glucose, N-acetylhexosamine, threonine and plant protein hydrolysates as monosaccharides, and Korean Patent No. 2222953 discloses a carbon source As a medium containing fructose and lactose, N-acetylhexosamine, L-aspartic acid, L-cysteine and plant peptone, etc. are proposed, but the production yield is high and economic medium for culturing Akkermansia genus microorganisms has been developed. The demand for is still there.

Korean Patent No. 1809172 Korean Patent No. 1799829 U.S. Patent No. 10,988,809 Korean Patent No. 2222953

Derrien et al., “Akkermansia muciniphila gen. nov., sp. nov., a human intestinal mucin-degrading bacterium” International Journal of Systematic and Evolutionary Microbiology (2004), 54, 1469-1476

The present invention is a microorganism of the genus Akkermansia that can be economically mass-produced with high production yield so that the microorganism of the genus Akkermansia can be industrially used as a medicine such as a microbiome treatment, food or feed for improving or preventing various diseases, In particular, it is to provide a culture medium composition for culturing Ackermansia municipilla and a culture method using the same, and also to provide a method for producing a freeze-dried powder of a microorganism of the genus Acamancia with improved survival rate and stability prepared using the same.

The present invention relates to a medium composition for culturing microorganisms of the genus Akkermansia containing N-acetylhexosamine and L-threonine, wherein the medium composition contains disaccharide as a carbon source and animal protein hydrolysate and yeast extract as a nitrogen source. It relates to a culture medium composition for culture of microorganisms belonging to the genus Akkermansia comprising.

The disaccharide is lactose, and the lactose may be the only carbon source in the medium composition.

The animal protein hydrolyzate may be tryptone.

The medium composition may further include cobalamin.

The medium composition contains 0.5-5 g/L of N-acetylhexosamine, 1-5 g/L of L-threonine, 5-20 g/L of yeast extract, 10-20 g/L of lactose, and 10-30 g of tryptone. /L, 0.01-1 mg/L of cobalamin and 0.2-1 g/L of L-cysteine.

The medium composition contains monobasic potassium phosphate 0.2-0.8 g/L, dibasic sodium phosphate 0.3-0.9 g/L, ammonium chloride 0.1-0.6 g/L, magnesium chloride 0.05-0.4 g/L, calcium chloride 0.05-0.4 g/L. L, it may further include 2-8 g/L of sodium hydrogen carbonate.

In addition, the present invention relates to a method for culturing microorganisms of the genus Akkermansia in which the culture medium composition is inoculated with microorganisms of the genus Akkermansia and cultured under anaerobic conditions.

The Akkermansia genus microorganism may be Akkermansia muciniphila.

In addition, the present invention comprises the steps of inoculating a microorganism of the genus Akkermansia in the culture medium composition and culturing under anaerobic conditions; Recovering microbial cells of the genus Akkermansia from the culture solution of the culturing step; any one sugar or sugar alcohol selected from sucrose, maltose and trehalose; and mixing a cryoprotection composition containing any one organic acid selected from propionic acid, acetic acid and butyric acid with the microorganism cells of the genus Akkermansia; And freeze-drying the mixture of the microbial cells, sugar or sugar alcohol and organic acid; relates to a method for producing a freeze-dried powder of microorganisms belonging to the genus Akkermansia, including.

In the cryoprotection composition, the sugar or sugar alcohol may include 1 to 40% by weight, the organic acid to 0.1 to 20% by weight, and the balance water.

30-300 parts by weight of the cryoprotective composition may be included with respect to 100 parts by weight of the Akkermansia microbial cells.

A microorganism of the genus Akkermansia of the present invention, in particular, a culture medium composition for culturing Akkermansia municipilla and a culture method using the same can be used industrially as a medicine such as a microbiome treatment, food or feed for improving or preventing various diseases , The production yield is high enough to reach a cell density of 2 X 10 ¹⁰ CFU / mL or more, and economical mass production is possible, and the number of viable cells of Akkermansia genus microorganisms measured by the plate counting method is also By increasing the survival rate and stability of Akkermansia genus microorganisms through the manufacturing method, it can be widely used in medicine, lactic acid bacteria preparations, dairy products and probiotics.

Figure 1 shows the absorbance according to the incubation time and the viable cell count at 24 hours ( It is a graph confirming CFU/mL).
Figure 2 is a comparison of the survival rate, that is, stability, according to the storage period of Preparation Example 4-4 prepared by adding propionic acid with sucrose in Experimental Example 4 and lyophilized with Preparation Example 4-2 prepared by adding only sucrose and freeze-dried it's a graph

The present invention is a medium composition for culturing microorganisms of the genus Akkermansia containing N-acetylhexosamine and L-threonine, wherein the medium composition contains disaccharide as a carbon source, and animal protein hydrolysate and yeast extract as a nitrogen source. It relates to a medium composition for culturing microorganisms belonging to the genus Akkermansia, which has a high production yield and is economical.

As used herein, the term "medium" or "culture medium" refers to a solid, semi-solid or liquid medium containing all nutrients and essential physical growth parameters necessary for microbial growth or proliferation.

Also, the term "cultivation" or "growth" of a microorganism means to increase the microbial organism by propagating it in a given culture medium under conditions conducive to its growth.

In the medium composition, N-acetylhexosamine is widely used as a substitute for mucin in culture of microorganisms belonging to the genus Ackermansia, and the N-acetylhexosamine is N-acetylglucosamine (Glc-NAc) and N-acetylgal It can be selected from lactosamine (Gal-NAc), preferably N-acetylglucosamine. In the medium composition, N-acetylhexosamine may be included in an amount of 0.5-5 g/L, preferably 1-3 g/L, and more preferably 1.5-2 gL. The medium composition of the present invention is economically viable in mass production because it can be produced in high yield while reducing the use of expensive N-acetylhexosamine used as a substitute for mucin.

Since mucin that can be degraded by microorganisms of the genus Akkermansia is composed of amino acid sequences rich in serine, threonine, proline and cysteine, when culturing microorganisms of the genus Akkermansia in a mucin-free medium, additional supply of these amino acids produces production It is advantageous to increase yield. Among the above amino acids, the supply of threonine is particularly important, and among these, it is very important to supply a combination of threonine and cysteine. In the medium composition, L-threonine may be included at 1-5 g/L, preferably at 1.2-4 g/L, and more preferably at 1.5-3 g/L. It may also contain cysteine at 0.2-1 g/L, preferably at 0.3-0.8 g/L, and more preferably at 0.3-0.6 g/L.

In addition to the above amino acids, alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, hydroxyproline, tryptophan, tyrosine, and valine can be additionally supplied. The amino acids may also be included in the medium composition at 0.05-0.5 g/L, if necessary. All of the above amino acids are preferably L-forms, and aspartic acid or cysteine may be D-forms.

According to one embodiment of the present invention, the disaccharide is lactose, and the lactose may be the only carbon source in the medium composition. In the medium composition of the present invention, using lactose alone as the sole carbon source is advantageous in increasing production yield compared to using glucose alone or in combination with other carbon sources as the carbon source. In the medium composition, lactose, which is the sole carbon source, may be included at 10-20 g/L, preferably 11-18 g/L, and more preferably 12-16 g/L.

According to another embodiment of the present invention, in the medium composition, animal protein hydrolysates and yeast extracts are used in combination as a nitrogen source. The animal protein hydrolyzate may be tryptone. The yeast extract may be yeast autolysate, ultrafiltered yeast extract or synthetic yeast extract.

Compared to using plant peptone such as soy peptone with yeast extract as a nitrogen source, using animal protein hydrolysate obtained by hydrolyzing casein, a milk protein, with trypsin, that is, tryptone in combination with yeast extract is advantageous in increasing production yield. When lactose is used as a carbon source, it is more significantly advantageous to increase production yield to use a combination of tryptone and yeast extract as a nitrogen source. In the medium composition, the animal protein hydrolyzate may be included at 10-30 g/L, preferably 11-25 g/L, and more preferably 12-20 g/L, and the yeast extract at 5-20 g/L , preferably 7-18 g/L, more preferably 9-16 g/L.

According to another embodiment of the present invention, the medium composition may further include cobalamin. In the medium composition, cobalamin may be included in an amount of 0.01-1 mg/L, preferably 0.05-0.5 mg/L, and more preferably 0.08-0.12 mg/L.

According to another embodiment of the present invention, the medium composition contains 1-5 g/L of N-acetylhexosamine, 1-5 g/L of L-threonine, 5-20 g/L of yeast extract, and 10-20 g of lactose. /L, tryptone 10-30 g/L, cobalamin 0.001-1 mg/L, and L-cysteine 0.2-1 g/L.

According to another embodiment of the present invention, the medium composition contains 0.2-0.8 g/L of potassium phosphate monobasic, 0.3-0.9 g/L of sodium phosphate dibasic, 0.1-0.6 g/L of ammonium chloride, and 0.05-0.4 g of magnesium chloride. /L, calcium chloride 0.05-0.4 g/L, and sodium bicarbonate 2-8 g/L may be further included.

According to another embodiment of the present invention, the medium composition contains 15 g/L of hydrolysed casein protein, 10 g/L of yeast extract, 2 g/L of L-threonine, and lactose. (lactose) 13.512 g/L, N-acetylhexosamine 1.659 g/L, Monopotassium Phosphate [KH2PO4] 0.4 g/L, Sodium Phosphate FG [Na2HPO4] 0.53 g/L, Ammonium chloride Ammonium Chloride [NH4Cl] 0.3 g/L, Magnesium Chloride [MgCl2] 0.1 g/L, Cacium Chloride [CaCl2] 0.11 g/L, Sodium Bicarbonate [NaHCO3] 4 g/L, Cyanocobalamin 0.0001 g/L, L-Cysteine, 0.5 g/L.

The medium composition may be a liquid medium as well as a powder or concentrated liquid medium. In this case, it can be dissolved in a predetermined volume of water, usually sterile water, before use.

In addition, the present invention relates to a method for culturing microorganisms of the genus Akkermansia in which the medium composition is inoculated with microorganisms of the genus Akkermansia and cultured under anaerobic conditions.

According to one embodiment of the present invention, the microorganism of the genus Akkermansia may be Akkermansia muciniphila.

The culturing may be carried out under conditions of pH 6.0 to 8.0, culture temperature of 35 to 40° C., stirring speed of 20 to 120 rpm, and nitrogen saturation of 100%.

According to the present invention, when cultured using the medium composition of the present invention, the production yield is excellent enough to reach a cell density of 2 X 10 ¹⁰ CFU / mL or more, as measured by the plate counting method.

In addition, the present invention comprises the step of inoculating a microorganism of the genus Akkermansia in the culture medium composition and culturing under anaerobic conditions; Recovering microbial cells of the genus Akkermansia from the culture solution of the culturing step; any one sugar or sugar alcohol selected from sucrose, maltose and trehalose; and mixing a cryoprotection composition containing any one organic acid selected from propionic acid, acetic acid and butyric acid with the microorganism cells of the genus Akkermansia; And freeze-drying the mixture of the microbial cells, sugar or sugar alcohol, and organic acid; relates to a method for producing a freeze-dried powder of microorganisms belonging to the genus Akkermansia, including.

In the cryoprotection composition, the content of sugar or sugar alcohol is 1-40% by weight, preferably 5-30% by weight, more preferably 7-20% by weight, and the organic acid is 0.1-20% by weight, preferably It may contain 1 to 15% by weight, more preferably 3-10% by weight, and may include other cryoprotective components as needed, the balance including water.

Based on 100 parts by weight of the Akkermansia microbial cells, the cryoprotection composition may include 30-300 parts by weight, preferably 50 to 150 parts by weight.

The sugar or sugar alcohol is preferably sucrose, and the organic acid may be preferably propionic acid.

The freeze-dried powder of Akkermansia genus microorganisms produced by the above method has a viability of 40% or more compared to the sample before freeze-drying. The survival rate is expressed as a percentage of the number of viable bacteria relative to the total number of bacteria. In addition, the stability measured after storage at room temperature for 6 months or more, preferably 1 year after production from the above method is 90% or more compared to the initial viable cell count.

Hereinafter, the present invention will be described in more detail through examples, but the present invention is not limited thereto.

[Production Example 1]

Media compositions of Preparation Examples 1-1 to 1-3 were prepared as shown in Table 1 below. Preparation Examples 1-1 and 1-3 are media using glucose as the sole carbon source, Preparation Example 1-2 is a medium using lactose by replacing glucose as the sole carbon source, Preparation Examples 1-1 and 1-2 are yeast as a nitrogen source Soy peptone, a plant peptone, was used together with the extract, and in Preparation Examples 1-3, tryptone, an animal protein hydrolyzate, was used instead of plant peptone.

ingredient 1-1 1-2 1-3 Soy Peptone 15g/L 15g/L - tryptone - - 15g/L L-Threonine 2g/L 2g/L 2g/L D-glucose (anhydrous) 4.512 g/L - 4.512 g/L lactose - 4.512 g/L - N-Acetylglucosamine 0.553 g/L 0.553 g/L 0.553 g/L Monopotassium Phosphate FG[KH ₂ PO _4] 0.4g/L 0.4g/L 0.4g/L Disodium Phosphate FG [Na2HPO4] 0.53g/L 0.53g/L 0.53g/L Sodium Chloride FG [NaCl] 0.3 g/L 0.3 g/L 0.3 g/L Magnesium Chloride FG [MgCl2] 0.1 g/L 0.1 g/L 0.1 g/L Cacium Chloride FG [CaCl2] 0.11g/L 0.11g/L 0.11g/L Sodium Bicarbonate FG [NaHCO3] 4g/L 4g/L 4g/L Vitamin B12 Crystalline (Cyanocobalamin) FG 0.1 mg/L 0.1 mg/L 0.1 mg/L L-Cittane Hydrochloride 0.5 g/L 0.5 g/L 0.5 g/L

[Experimental Example 1]

After inoculating the Ackermansia municipilla strain at a concentration of 1% (v / v) in the medium composition of Preparation Example 1-1 and Preparation Example 1-2, pH 7.0, culture temperature 37 ℃ stirring speed 80 rpm, nitrogen saturation It was cultured for 24 hours under 100% conditions.

As the Ackermansia municipilla strain, DSM 22959 strain (hereinafter referred to as 'HB01' strain), KCCM12318P strain (hereinafter referred to as HB03 strain) and KCCM12424P strain (hereinafter referred to as HB05 strain) possessed by the present applicant were used.

At 24 hours, the absorbance (600 nm OD) of the culture medium was measured and shown in Table 2, and the total number of bacteria (cell / mL) of the culture medium measured by the total number of bacteria count method using Automatic Cell counter (Tx Microbial Cell counter, Logos Biosystem) equipment It is measured and shown in Table 3, and the number of viable cells (CFU / mL) of the culture medium is measured by the plate counting method and shown in Table 4.

division OD at 600 nm Preparation Example 1-1 Production Example 1-2 HB01 strain 1.795±0.12 2.692± ^0.21a HB03 strain 1.931±0.18 2.795± ^0.24a HB05 strain 2.012±0.19 2.895± ^0.22b

a: Significant value as P<0.05 by student's t-test / b: No significant value In order to determine statistical significance according to the difference in medium in the same strain, use the student's t-test method Only when the p value was less than 0.05 was determined to be statistically significant, and the same significant difference was indicated below.

division Total bacterial count (X10 ⁹ cell/mL) Preparation Example 1-1 Production Example 1-2 HB01 strain 5.75±0.25 6.91± ^0.28b HB03 strain 6.03±0.28 8.32± ^0.32b HB05 strain 6.12±0.31 8.28± ^0.30b

division Viable cell count (X10 ⁹ CFU/mL) Preparation Example 1-1 Production Example 1-2 HB01 strain 1.84±0.09 2.52± ^0.12b HB03 strain 2.89±0.15 4.02±0.13 ^a HB05 strain 3.07±0.16 4.41± ^0.18b

Compared to Preparation Example 1-1 using glucose as the sole carbon source in all three Ackermansia municipilla strains, the medium composition of Preparation Example 1-2 using lactose tends to increase both absorbance, total bacterial count and viable cell count showed up

However, in Preparation Example 1-2 using lactose, compared to Preparation Example 1-1 using glucose, only the HB05 strain significantly increased the absorbance, and the total number of bacteria significantly increased all strains in all strains HB01, HB03 and HB05 , The number of viable cells significantly increased only in strains HB01 and HB05, showing differences depending on the type of Ackermansia municipilla strain.

[Experimental Example 2]

After inoculating the Ackermansia municipilla strain at a concentration of 1% (v / v) in the medium composition of Preparation Example 1-1 and Preparation Example 1-3, pH 7.0, culture temperature 37 ℃ stirring speed 80 rpm, nitrogen saturation It was cultured for 24 hours under 100% conditions.

As the Ackermansia municipilla strain, DSM 22959 strain (hereinafter referred to as 'HB01' strain), KCCM12318P strain (hereinafter referred to as HB03 strain) and KCCM12424P strain (hereinafter referred to as HB05 strain) possessed by the present applicant were used.

At 24 hours, the absorbance (600 nm OD) of the culture medium was measured and shown in Table 5, and the total number of bacteria (cell / mL) of the culture medium measured by the total number of bacteria count method using Automatic Cell counter (Tx Microbial Cell counter, Logos Biosystem) equipment It is measured and shown in Table 6, and the number of viable cells (CFU / mL) of the culture medium is measured by the plate counting method and shown in Table 7.

division OD at 600nm Preparation Example 1-1 Production Example 1-3 HB01 strain 1.872±0.14 2.432± ^0.18b HB03 strain 1.973±0.20 2.782± ^0.22a HB05 strain 1.997±0.19 2.885±0.24 ^a

division Total bacterial count (X10 ⁹ cell/mL) Preparation Example 1-1 Production Example 1-3 HB01 strain 5.31±0.21 7.12± ^0.29b HB03 strain 5.99±0.30 8.08± ^0.35b HB05 strain 6.01±0.28 8.12± ^0.36b

division Viable cell count (X10 ⁹ CFU/mL) Preparation Example 1-1 Production Example 1-3 HB01 strain 1.75±0.12 2.65± ^0.14a HB03 strain 2.15±0.14 3.85± ^0.18a HB05 strain 2.75±0.14 3.98±0.20 ^a

Compared to Preparation Example 1-1 using plant peptone as a nitrogen source in all three Ackermansia municipilla strains, 1-3 using tryptone as a nitrogen source showed a tendency to increase absorbance, total bacterial count, and viable cell count. .

However, compared to Preparation Example 1-1 using glucose in Preparation Example 1-2 using lactose, only the HB01 strain significantly increased the absorbance, and the total number of bacteria significantly increased all strains in all strains HB01, HB03 and HB05 , The number of viable cells did not show a significant increase in all strains of HB01, HB03 and HB05, indicating differences depending on the type of Ackermansia municipilla strain.

[Production Example 2]

Media compositions of Examples and Comparative Examples 1 and 2 were prepared as shown in Table 8 below. Example uses lactose by replacing glucose as the sole carbon source in the basal medium of Preparation Example 1-1, uses tryptone, which is an animal protein hydrolyzate, and yeast extract as nitrogen sources, and uses L-threonine and L-threonine as amino acids. cysteine was added.

Comparative Example 1 is one of the conventionally known culture medium compositions of Ackermansia municipilla, using glucose as the sole carbon source, using soy peptone as the nitrogen source, and adding L-threonine as an amino acid. is a composition

Comparative Example 2 also uses a combination of fructose and lactose as a carbon source as one of the previously known media compositions for culturing Ackermansia municipilla, and also uses a combination of plant peptone and yeast extract as a nitrogen source, and uses L-aspartic acid and L-aspartic acid as amino acids. It is a medium composition characterized by the addition of L-cysteine.

ingredient Comparative Example 1 Comparative Example 2 Example Soy Peptone 16g/L 20g/L - tryptone - - 15g/L yeast extract - 10 g/L 10 g/L L-Threonine 4g/L - 2 g/L L-cysteine - 0.5 g/L 0.5 g/L L-aspartic acid - 8 g/L - D-glucose (anhydrous) 4.504g/L - - fructose - 2.5g/L - lactose - 2.5 g/: 13.512 g/L N-Acetylglucosamine 5.53025g/L 5g/L 1.659 g/L KH ₂ PO ₄ 0.4g/L - 0.4g/L K2HPO4 - 2.5g/L - Na2HPO4 0.53g/L - 0.53g/L NH4Cl 0.3 g/L - 0.3g/L NaCl 0.3 g/L - - MgCl2.6H2O 0.1 g/L - 0.1 g/L CaCl2 0.11g/L - 0.11g/L alkaline trace element solution 1ml/L - - acid trace element solution 1ml/L - - vitamin solution 1ml/L - - Vitamin B12 Crystalline (Cyanocobalamin) FG - 0.1 mg/L 0.1 mg/L Resazurin 0.5 mg/L - - NaHCO3 4g/L - 4g/L Na2S.7-9H2O. 0.25 g/L - -

[Experimental Example 2]

After inoculating the Ackermansia municiphila strain at a concentration of 1% (v / v) in the medium composition of Comparative Examples 1 and 2, Example of Preparation Example 2, pH 7.0, culture temperature 37 ° C. stirring speed 80 rpm, nitrogen saturation It was cultured for 24 hours under 100% conditions. As the Ackermansia municipila strains, HB01, HB03 and HB05 strains were used in the same manner as in Experimental Example 1.

The total bacterial count (cell / mL) of the culture medium measured by the total bacterial count method using the Automatic Cell counter (Tx Microbial Cell counter, Logos Biosystem) equipment is measured and shown in Table 9, and the viable cell count (CFU / mL) was measured and shown in Table 10.

division Total bacterial count (X10 ⁹ cell/mL) Comparative Example 1 Comparative Example 2 Example HB01 strain 7.93±0.41 0.70±0.17 11.00±0.68 ^a,b HB03 strain 6.43±0.45 1.75±0.18 15.30±0.51 ^a,b HB05 strain 5.89±0.32 1.59±0.15 13.50±0.49 ^a,b

division Viable cell count (X10 ⁹ CFU/mL) Comparative Example 1 Comparative Example 2 Example HB01 strain 1.10±0.15 0.00±0.00 4.50±0.21 ^a,b HB03 strain 1.05±0.11 0.0020±0.0003 9.10±0.56 ^a,b HB05 strain 1.05±0.16 0.0018±0.0002 9.78±0.62 ^a,b

a or b: significant value as P<0.05 by student's t-test / c: no significant value In order to determine statistical significance according to the difference in medium in the same strain, the student's t-test method was used It was determined that it was statistically significant only when the p value was less than 0.05, and cases where the p value was less than 0.05 in Examples compared to Comparative Examples 1 and 2 were indicated as a and b, respectively.

In Preparation Example 1-2 in which only glucose was changed to lactose in the basal medium of Preparation Example 1-1 and Preparation Example 1-3 in which soy peptone was changed to tryptone, there was a significant difference in the increase in total strains and viable cell counts. Although differences were shown depending on the type of Sipilla strain, in the medium of Example, a significant increase in the total number of strains and viable cells was confirmed in all strains compared to the medium of Comparative Examples 1 and 2, and the amount was at least 2 It was confirmed that the production yield was significantly increased compared to the conventional medium by more than two times.

[Experimental Example 3]

The experiment of Experimental Example 3 was conducted to confirm whether the medium composition of Example exhibits a yield increase effect compared to the production medium composition of Comparative Examples 1 and 2 when cultured by scale-up in a 10 L fermentor.

In a 10L fermenter, each 7L of the medium composition of Comparative Examples 1 and 2 and Example of Preparation Example 2 was inoculated with the Ackermansia municophila strain at a concentration of 1% (v / v), pH 7.0, culture temperature 37 ℃ stirring rate It was cultured for 48 hours at 80 rpm and 100% nitrogen saturation. As the Akkermansia municipila strain, HB03 strain was used.

The absorbance (OD) was measured at 600 nm every hour, and the number of viable cells (CFU/mL) of the culture medium at 24 hours was measured by plate counting method, and is shown in FIG. 1 . At 24 hours, the number of viable cells in each culture was 2.02 X 10 ¹⁰ CFU/mL in Example, 1.02 X 10 ¹⁰ CFU/mL in Comparative Example 1, and 4.12 X 10 ⁹ CFU/mL in Comparative Example 2. The medium composition of Example showed a difference in the number of viable cells by about 20 times compared to Comparative Example 1 and about 2 times or more compared to Comparative Example 2.

[Experimental Example 4]

Ackermansia municipilla strain (strain HB03) of Experimental Example 3 At 24 hours, the culture medium was transferred to an anaerobic chamber under anaerobic conditions, and the cells were recovered through a Tangential Flow Filtration (TFF) system. After concentrating only the cells through the TFF system, the concentrated HB03 strain cells were collected in a sterilized bottle. 100 parts by weight of the cells were mixed with 80 parts by weight of the cryoprotective composition dissolved in purified water while changing the contents of sucrose and propionic acid, and then each sample was sealed with a vacuum packaging machine. The sealed vacuum bag was taken out of the chamber, pre-frozen in a deep freezer (-70 ° C), and freeze-dried by applying a vacuum and freezing for 24 hours.

The total number of bacteria and the number of viable cells in the freeze-dried sample were measured by the method described in Experimental Example 1, and the value obtained by dividing the number of viable cells by the total number of bacteria in % was defined as the viability and is shown in Table 11.

division 4-1 4-2 4-3 4-4 4-5 Sucrose (% by weight) 3.0 6.0 3.0 6.0 8.8 Propionic acid (% by weight) 0.0 0.0 5.0 5.0 5.0 Viable cell count (X 10 ¹¹ CFU/g) 5.46±0.72 6.99±0.88 8.58±0.82 6.97±1.01 6.70±0.34 Total bacterial count (X 10 ¹¹ cell/g) 37.35±1.43 39.10±1.68 23.70±1.23 16.20±2.45 13.80±1.21 survival rate (%) 13.39±1.55 17.89±1.45 36.30±2.78 ^a 42.98
± ^2.67b 48.79± ^1.85b

In order to determine the statistical significance of Preparation Examples 4-2 to 4-5 compared to Preparation Example 4-1, the student's t-test method is used to indicate a if the p value is less than 0.05, and the p value is less than 0.005 In case b, it was judged to be statistically significant. When the weight of sucrose was increased by 2 times compared to Preparation Example 4-1, the survival rate showed a tendency to increase slightly, but it was not statistically significant. However, when propionic acid was added compared to Preparation Example 4-1, the survival rate significantly increased more than twice (p<0.05).

Preparation Examples 4-4 and 4-5 in which the content of sucrose was increased to 6.0% by weight and 8.8% by weight compared to Preparation Example 4-3 showed a tendency to increase the survival rate more, and the survival rate compared to Preparation Example 4-1 This significantly increased more than 3 times (p<0.005).

The viability was measured while the samples of Preparation Examples 4-2 and 4-4 were stored at room temperature (20 ° C), and the change in relative survival rate according to the storage period was calculated as stability based on the initial survival rate, and it is shown in FIG. .

According to FIG. 2, in the case of the sample of Preparation Example 4-2, the survival rate after 6 months from the initial survival rate decreased by 47%, and in the case of the sample of Preparation Example 4-4, the survival rate decreased by only 8% after 6 months from the initial survival rate. Storage stability was remarkably excellent when lozose and propionic acid were combined.

Claims (11)

In the medium composition for culturing microorganisms of the genus Akkermansia containing N-acetylhexosamine and L-threonine,
The medium composition is a medium composition for culturing microorganisms belonging to the genus Akkermansia comprising disaccharides as a carbon source, and animal protein hydrolysates and yeast extracts as nitrogen sources. According to claim 1, wherein the disaccharide is lactose, the medium composition for culturing Akkermansia genus microorganisms, characterized in that the only carbon source in the medium composition. The medium composition for culturing microorganisms belonging to the genus Akkermansia according to claim 1, wherein the hydrolyzate of animal protein is tryptone. According to claim 1, wherein the medium composition for culturing Akkermansia genus microorganisms characterized in that it further comprises cobalamin. The method of claim 4, N-acetylhexosamine 0.5-5 g/L, L-threonine 1-5 g/L, yeast extract 5-20 g/L, lactose 10-20 g/L, tryptone 10- 30 g / L, cobalamin 0.01-1 mg / L and L- cysteine 0.2-1 g / L characterized in that it comprises a medium composition for culturing Akkermansia genus microorganisms. The method of claim 5, wherein the medium composition contains monobasic potassium phosphate 0.2-0.8 g/L, dibasic sodium phosphate 0.3-0.9 g/L, ammonium chloride 0.1-0.6 g/L, magnesium chloride 0.05-0.4 g/L, 0.05-0.4 g/L of calcium chloride and 2-8 g/L of sodium bicarbonate A medium composition for culturing Akkermansia genus microorganisms. A method for culturing microorganisms of the genus Akkermansia in which the microorganisms of the genus Akkermansia are inoculated and cultured under anaerobic conditions in the culture medium composition according to any one of claims 1 to 6. According to claim 7, wherein the Akkermansia genus microorganism is Akkermansia muciniphila ( Akkermansia muciniphila ) Method for culturing a microorganism of the genus Akkermansia, characterized in that. Claims 1 to 6 of any one of the culture medium composition Akkermansia ( Akkermansia ) Step of inoculating a microorganism of the genus and culturing under anaerobic conditions;
Recovering microbial cells of the genus Akkermansia from the culture solution of the culturing step;
any one sugar or sugar alcohol selected from sucrose, maltose and trehalose; and mixing a cryoprotection composition containing any one organic acid selected from propionic acid, acetic acid and butyric acid with the microorganism cells of the genus Akkermansia; and
Freeze-drying the mixture of the microbial cells, sugar or sugar alcohol and organic acid; A method for producing a freeze-dried powder of microorganisms belonging to the genus Akkermansia comprising a. The method according to claim 9, wherein the cryoprotective composition comprises 1 to 40% by weight of the sugar or sugar alcohol, 0.1 to 20% by weight of the organic acid, and the balance of water. The method according to claim 9, wherein the cryoprotective composition comprises 30-300 parts by weight based on 100 parts by weight of the Akkermansia microbial cells.

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