KR20200076450A - Bacteria having increased glycocalyx and method for cultivating the same - Google Patents

Abstract

The present invention relates to a method of culturing a strain with increased formation of glycocalyx. More particularly, the present invention relates to a method of culturing a Rhodobacter capsulatus strain, a Rhodobacter blasticus strain, or a mixed strain thereof using a medium where malic acid is used as a carbon source.

Description

Bacterial HAVING INCREASED GLYCOCALYX AND METHOD FOR CULTIVATING THE SAME

The present invention relates to a method for culturing a strain having an increased glycolytic calix, and to establishing a culture condition for a bacterium having an increased formation of the glyco calix.

Glico Calix performs functions such as adhesion to host cells, nutrients, prevention of drying, and protection of cells from the host immune system, and is a linear protein composed of hydrocarbons bound to lipids present in the cell membrane and membrane-binding sugars. It refers to oligosaccharides such as proteins.

Glyco Calyx, which is produced by microorganisms, has a wide range of functions such as emulsification stability, surface tension control, water absorption, adhesion, lubrication, and biofilm formation, depending on conditions, and is used as a material for various industries such as the medical industry. Is becoming. In particular, the microbial glycocolic can improve productivity by improving production culture conditions with high potential for industrial use, and mass production by continuous culture using a fermentation tank in a short period of time is possible. Easy separation and recovery.

Therefore, research on these microorganisms and microalgal glycocoli is actively being conducted, and for example, PCT application patent WO2018-110739 A1 extracts glycocoli from Rhodobacter capsulatus and Rhodobacter blasticus strains. Although a method of producing is disclosed, the production amount of the glycos calix of the patent is a dry weight of up to 0.25 g/l, and the production amount of the glycos calix is difficult to meet industry expectations.

Therefore, it is expected that the strain can be widely applied in related fields when a strain having an increased formation of a glycocalis having a wide range of utilization is provided.

One aspect of the present invention is to provide a method for culturing a strain in which the formation of glycocoli is increased.

According to one aspect of the present invention, a method for culturing a Rodobacter capsulatus strain, a Rodobacter blasticus strain, or a mixed strain thereof using a medium using malic acid as a carbon source is provided.

According to the present invention, as a method for culturing a strain having an increased amount of formation of a glycos calix is provided, the culture conditions of the glycos calix are improved to enable mass production of the glycos calix, thereby enabling industrial use of the glycos calix. You can meet your expectations.

In Figure 1 (a) is in the method of analyzing the neutral sugar in the Examples of the present invention, (b) in the method of analyzing the acidic sugar in the Examples of the present invention, (c) in the method of analyzing the protein in the Examples of the present invention It represents the prepared standard solution (STD), and represents a solution in which each STD and each STD and the glycocalis membranes of each strain (cS, cM, bS and bM) are mixed.
2A to 2D show the analysis results of analytical chromatography per component of the glyco calix (cS, cM, bS and bM) of each strain in the embodiment of the present invention. 2A shows cS, FIG. 2B shows cM, FIG. 2C shows bS, and FIG. 2D shows analysis results of bM.
3A to 3D show measurement results of molecular weight measurement chromatography of glyco calix (cS, cM, bS, and bM) of each strain in Examples of the present invention. 3A shows the analysis results of cS, FIG. 3B shows cM, FIG. 3C shows bS, and FIG. 3D shows bM.
Figure 4 shows the extraction process of the glyco calix of each strain in the embodiment of the present invention.
Figure 5 shows the appearance change of the culture medium according to the culture age change, when culturing each strain in the embodiment of the present invention.

Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. However, the embodiments of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below.

According to the present invention, there is provided a method for culturing a strain in which the formation of glycocoli is increased.

The strain forming the glycocoli of the present invention may be a strain of Rhodobacter capsulatus, a strain of Rhodobacter blasticus, or a mixture thereof.

The Rhodobacter capsulatus strain and the Rhodobacter blasticus strain were purchased from the Environmental Microbiology Bank (KEMB).

In more detail, the culture method of the present invention is a method of culturing Rhodobacter capsulatus strain, Rhodobacter blasticus strain, or a mixed strain thereof using a medium using malic acid as a carbon source.

The carbon source may further include carbon sources such as succinic acid, fructose, glucose, and lactic acid together with malic acid, but it is preferable to include malic acid alone.

The medium that can be applied to the present invention includes ferric citrate solution, KH ₂ PO ₄ , MgSO ₄ ㆍ7H ₂ O, NaCl, NH ₄ Cl, CaCl ₂ ㆍ2H ₂ O and trace element solution ) It is preferred to further include SL-6.

For example, the medium is 1.0 ml of ferric citrate solution (0.5%) per liter of distilled water, 0.5 g of KH ₂ PO ₄ 0.5 g, MgSO ₄ ㆍ7H ₂ O 0.4 g, NaCl 0.4 g, NH ₄ Cl 0.4 g , CaCl ₂ ㆍ2H ₂ O 0.05 g and trace element solution SL-6 containing 1.0 ml, and using a medium having a pH of 6.8.

Meanwhile, the medium preferably contains the carbon source in an amount of 0.01 to 0.1 wt% based on the weight of the entire medium, and is preferably 0.05 to 0.1 wt%. More preferably, it is contained in an amount of 0.1 wt%.

When the carbon source is less than 0.01wt% based on the weight of the entire medium, the strain growth rate is lowered, and when it exceeds 0.1wt%, the production amount of glyco calix is not significantly different, so economic efficiency is deteriorated.

The culture of the present invention is preferably carried out at pH 6.5 to 7, when the pH is too high or low, the metabolism of bacteria decreases and the cell activity decreases, so that the growth rate of the strain and the production rate of glycocalis are reduced.

Inoculation of the strain to the medium is preferably inoculated with 0.5 to 5 (v/v)% of the strain on a medium-by-volume basis, more preferably 1 to 2 (v/v)% of the strain. will be. When the inoculation amount is less than 0.5 (v/v)%, there is a problem that the strain growth rate is lowered, and when it exceeds 5 (v/v)%, the formation amount of glyco calix is not significantly different, and thus economic efficiency is deteriorated.

It is preferable that the culture of Rhodobacter capsulatus strain, Rhodobacter blasticus strain, or a mixed strain thereof is carried out under aerobic anaerobic conditions.

On the other hand, the culture of the present invention is preferably carried out at 25 to 37 ℃, more preferably carried out at 25 to 30 ℃, if the temperature is less than 25 ℃ there is a problem that the continuous growth and activity of the strain is inhibited In the case of exceeding 37 ℃, there is a problem that the growth rate is reduced due to the high temperature.

According to the present invention, at least 3.5 g/L of glyco calix is formed per 1 L of the volume of the medium, preferably at least 3.7 g/L of glyco calix is formed, and in particular, Rhodobacter encapsulation using carbon source as malic acid. When the strain was cultivated, a significantly increased amount of glyco calix exceeding 9 g/L is formed.

Hereinafter, the present invention will be described in more detail through specific examples. The following examples are only examples for helping the understanding of the present invention, and the scope of the present invention is not limited thereto.

Example

1. Preparation of Glico Calix

1) Acquisition of strain

As a strain forming Glyco calix, the Rhodobacter capsulatus strain and the Rhodobacter blasticus strain were obtained from the Environmental Microbial Bank (KEMB).

2) Cultivation of Rhodobacter capsuleus strain and Rhodobacter blasticus strain

The culture conditions for culturing each strain were inoculated with 1% (v/v) of bacteria in a liquid medium, and cultured at 28°C for 30 days under a permeable anaerobic condition. The liquid medium composition is 1.0 g of yeast extract per liter of distilled water, 1.0 g of disodium succinate hexahydrate, 0.5 ml of absolute ethanol, 1.0 ml of ferric citrate solution, 0.5% , KH ₂ PO ₄ 0.5 g, MgSO ₄ ㆍ7H ₂ O 0.4 g, NaCl 0.4 g, NH ₄ Cl 0.4 g, CaCl ₂ ㆍ2H ₂ O 0.05 g and trace element solution SL-6 1.0 ml And adjusted to PH 6.8 with 1 N NaOH.

5 shows the change in appearance of the culture medium according to the culture age change of the culture medium 5L during the culture of the strain. In the upper and lower diagrams of (a) to (c), the medium on the left is the appearance of the culture medium of Rhodobacter capsuleus strain, and the medium on the right is the appearance of the culture medium of Rhodobacter blastitis strain. In addition, (a) is 2 days, (b) is 7 days, and (c) is 14 days old culture.

3) Formation of glycocoli according to carbon source

In order to determine the production amount of glyco calix according to the carbon source, 1.0 ml of ferric citrate solution (0.5%) per liter of distilled water, 0.5 g of KH ₂ PO ₄ 0.5 g, MgSO ₄ ㆍ7H ₂ O 0.4 g, NaCl 0.4 g, NH ₄ Cl 0.4 g, CaCl ₂ ㆍ2H ₂ O 0.05 g, trace element solution SL-6 1.0 ml of each carbon source (Succinate, Malic acid) was added to 1.0 ml of inorganic medium, After adjusting the pH to 6.8, 1% (v/v) of bacteria was inoculated into the liquid medium and incubated at 25°C for 30 days under aerobic anaerobic conditions.

In Figure 5 (a) to (c), the upper figure of each figure shows the appearance of the culture medium when culturing succinic acid as a carbon source, and the lower figure shows the appearance of culture medium when culturing malic acid as a carbon source.

4) Extraction of glycocoli of each strain

5L of the culture medium cultured by the method described in 1.3) was concentrated using a mass concentrator, after which the concentrated culture medium was centrifuged at 1500 rpm for 30 minutes to separate cells, and the obtained supernatant was taken with ethanol 1: The mixture was mixed with 4 (v/v) and precipitated at 4° C. for 12 hours or more to separate Glyco Calyx. Then, the precipitate was separated by centrifugation at 1500 rpm for 10 minutes, and then dried using a freeze dryer.

The process of extracting glycos of each strain is shown in FIG. 5. (a) shows the process of concentrating the culture medium using a mass concentrator, (b) shows the concentrated culture medium, and (c) the culture solution and ethanol are 1:4 (v/v)% mixed solution. (D) shows the precipitated Glyco calix. In addition, (e) shows a process of drying the precipitated glycos calix using a freeze dryer, and (f) shows the glycos calix obtained by drying.

As a result, the obtained glyco calix was subjected to quantitative analysis of saccharides and proteins according to carbon sources and strains, analysis of constituent sugars, molecular weight measurement, and production of glyco calix.

Hereinafter, the glycocalix obtained by culturing the Rhodobacter capsulotus strain using succinic acid as a carbon source is referred to as cS, and the glycocalis obtained by culturing the Rhodobacter capsulotus strain using malic acid as a carbon source is referred to as cM, The glycocalix obtained by cultivating the Rhodobacter blasticus strain with succinic acid as a carbon source is referred to as bS, and the glycocalix obtained by culturing the Rhodobacter blasticus strain with malic acid as a carbon source is referred to as bM.

2. Analysis of the composition of Glico Calix according to the carbon source

(1) Method for quantitative analysis of saccharides and proteins of Glyco Calyx

1) Method and result of quantitative analysis of neutral sugar

Analysis method using phenol-sulfuric acid (Phenol-H ₂ SO ₄ ) It was analyzed by Phenol-H ₂ SO ₄ analysis method, and Galactose was used as a standard. All experiments were performed in 3 replicates. The specific analysis method is as follows.

① Prepare bovine serum albumin (BSA) and cS, cM, bS, and bM provided in 1. above at 1000 μg/mL.

② Dilute the BSA and the cS, cM, bS and bM 10 times.

③ STD preparation: Based on the diluted BSA and water, 0:200 (concentration 0 μL/mL), 40:160 (concentration 20 μL/mL), 80:120 (concentration 40 μL/mL), 120: A standard solution (STD) was prepared by mixing 80 (concentration 60 μL/mL), 160:40 (concentration 80 μL/mL) and 200:0 (concentration 100 μL/mL). The images of each STD are shown as S1, S2, S3, S4, S5, and S6 in FIG. 1(a).

④ In the test tube (glass), add 200 µL of each of the STD and the cS. cM, bS and bM are similarly added to the test tube with STD 200 μl.

⑤ Add 200 µl of 5% phenol to all tubes and vortex.

⑥ Sulfuric acid (H ₂ SO ₄ ) using a dispenser Add 1 ml each and vortex. At this time, the reaction is violent and heat is generated.

⑦ Leave it at room temperature for 30 minutes to cool the heat.

⑧ Using a pipette for sulfuric acid, add 200 µl of the solution of the tube performing the process of ⑦ to a 96-well plate. The image of the solution prepared in the process of ⑧ was shown as cS, cM, bS and bM in FIG. 1(a).

⑨ Measured at 490 nm with ELISA.

2) Method for quantitative analysis of acidic sugar

The analytical method is an analytical reagent prepared by dissolving sulfuric acid for uronic acid (H ₂ SO ₄ , 1.3 g sodium borate per 500 ml (Na ₂ B ₄ O ₇ ·10H ₂ O)-melting all day) and m-hydroxydiphenyl It was analyzed by the m-Hydroxydiphenyl analysis method using, Galacturonic acid (Galacturonic acid) was used as a standard. All experiments were performed in 3 replicates. The specific analysis method is as follows.

① Prepare bovine serum albumin (BSA) and cS, cM, bS, and bM provided in 1. above at 1000 μg/mL.

② Dilute the BSA and the cS, cM, bS and bM 10 times.

③ STD preparation: Based on the diluted BSA and water, 0:1000 (concentration 0 μL/mL), 200: 800 (concentration 20 μL/mL), 400:600 (concentration 40 μL/mL), 600: A standard solution (STD) was prepared by mixing 400 (concentration 60 μL/mL), 800:200 (concentration 80 μL/mL) and 1000:0 (concentration 100 μL/mL). The images of each STD are shown as S1, S2, S3, S4, S5, and S6 in FIG. 1(b).

④ The STD and the cS, cM, bS and bM are prepared in an E-tube.

⑤ After filling the Styrofoam box with ice, put a rack containing a test tube. (At this time, add a little water to facilitate the experiment. At this time, heat the water in advance and preheat ELISA in advance. Leave it on.)

⑥ Add 1.2 ml of the sulfuric acid for uronic acid to all test tubes (glass) using a dispenser.

⑦ Add 200 µL of STD and the cS to the E-tube placed in ice. cM, bS and bM are similarly added to the E-tube with STD 200 μl. After that, all E-tubes are vortexed. At this time, the reaction is violent and heat is generated.

⑧ Wrap the E-tube in step ⑦ with wrap and rubber band, put it in boiling water and simmer for 5 minutes. (Sleep is slightly higher than the scale of the E-tube.)

⑨ Immediately after removing the wrap, cool it in ice water. At this time, the E-tube has a light pink color.

확인 After confirming that it has cooled sufficiently, add 20 μl of m-hydroxydiphenyl and vortex. At this time, the color becomes a little darker.

⑪ After standing for 5 minutes, 200 µl of the solution of ⑩ is added to the 96-well plate using a pipette for sulfuric acid. The image of the solution prepared in the process of ⑪ is shown as cS, cM, bS, and bM of FIG. 1(b).

측정 Measured at 520nm with ELISA.

3) Quantitative analysis method of protein

The analysis method was analyzed through Bradford analysis method using Coomasie dye (Bio-rad), and bovine serum albumin (BSA, A2153-50G, Sigma) was used as a surface material. Did. All experiments were performed in 3 replicates. The specific analysis method is as follows.

① BSA and cS, cM, bS and bM provided in 1. are all made into 1000 μg/mL and diluted to 100 μg/mL.

② STD preparation: 0:200 (concentration 0 μL/mL), 40:160 (concentration 20 μL/mL), 80:120 (concentration 40 μL/mL), 120: A standard solution (STD) mixed with 80 (concentration 60 μL/mL), 160:40 (concentration 80 μL/mL) and 200:0 (concentration 100 μL/mL) was prepared. The images of each STD are shown as S1, S2, S3, S4, S5, and S6 in FIG. 1(c).

③ Add 200 μl of the STD and the cS to disposable plastic tubes. The cM, bS and bM are also put into disposable plastics at 200 μl with STD.

④ Put 50 μl of Coomassie die in all tubes and vortex.

⑤ After allowing to stand at room temperature for 5 minutes, add 200 µl of the solution from ④ to the 96-well plate. The photographed image of the solution prepared in the process of ⑤ is shown as cS, cM, bS and bM in FIG. 1(c).

⑥ Measure at 595 nm with ELISA.

Table 1 shows the analysis results by the method for quantitatively analyzing the neutral sugar, acidic sugar, and protein.

Strain name Carbon source Chemical composition (mol%) Cathedral Acidic sugar (Uronic acid) protein Rhodobacter Capsule Succinic acid 10.0±0.006 1.0±0.009 22.2±0.003 Malsan 8.0±0.007 1.8±0.006 12.5±0.009 Rhodobacter Blasticus Succinic acid 9.3±0.002 1.6±0.002 21.9±0.006 Malsan 5.4±0.002 1.2±0.002 11.3±0.005

As shown in Table 1, both the Rhodobacter capsulatus strain and the Rhodobacter blasticius strain produced glycocox proteins and neutral sugars in a ratio of about 2:1, and a small amount of acidic sugars. Was confirmed.

The composition of Glyco calix according to the strain did not differ significantly, but the Rhodobacter encapsulation strain was 0.7 mol% when succinic acid was used as a carbon source, and 2.6 mol% more neutral when malic acid was used. It was analyzed to be composed of sugar.

In addition, both strains used in the experiment contained more protein and neutrophils than the glyco calix produced using malic acid, and the glyco calix produced using succinic acid as a carbon source.

When malic acid was used as a carbon source in the Rhodobacter capsulotus strain, it was analyzed that the acidic sugar of the produced glycocalis was 0.8 mol% more than when succinic acid was used.

(2) Glyco Calix's compositional party analysis method

The analysis method was analyzed by using the method of adiitol acetate, and as standard, 7 types of monosaccharides (Rhamnose, Fucose, Arabinse, Xylose, Mannose ( Mannnose), Galactose, Glucose, Galacturonic acid, Glucuronic acid and Glucosamine were prepared at a concentration of 1 mg/mL. Prepared on the basis of 10mL and stored frozen, all experiments were carried out in duplicate. The specific analysis method is as follows.

① Add 1mg of sample (cS, cM, bS, and bM provided in 1.) to the test tube (glass) and then use 2M concentration of trifluoroacetic acid (TFA) to hydrolyze polysaccharide into monosaccharide. Add 1 ml and seal and heat in an oven at 121° C. for 90 minutes.

② When the reaction is over, cool the tube and dry it with nitrogen gas for 1 hour.

③ To remove the remaining TFA, add 200 ul of methanol and dry it using nitrogen gas for 10 minutes.

④ To open the ring structure of monosaccharides (opening process), add 350 ul of ammonia water (prepared by dissolving 50 mg of NaBH ₄ per 1 ml of NH ₄ OH at 1 M concentration) and 150 ul of NH ₄ OH at 1 M concentration for 4 hours at room temperature. To react.

⑤ To remove the remaining NaBH ₄ , add 10% acetic acid in 100 ul increments. Repeat this 6 times.

⑥ Dry for 2 hours using nitrogen gas.

⑦ To remove the remaining NaBH ₄ and acetic acid, add 1 ml of methanol and dry with nitrogen gas for 30 minutes. Repeat this 5 times.

⑧ Finally, after adding 1 ml of methanol, dry it thoroughly using nitrogen gas.

⑨ Immediately before use, add 20 ul of acetone to the dried sample and dissolve it for analysis.

The measurement method is measured using Gas Chromatography (GC) to which a Flame Ionization Detector (FID) is connected. Analysis results of analytical chromatography per component of Glyco Calyx according to the above method are shown in FIG. 2, (a) cS, (b) cM, (c) bS, (d) bM analysis results Indicates.

The results of chromatographic analysis per component of Glico Calix according to the above method are shown in Table 2 below.

Strain name Carbon source Per composition (mol%) Rhamnose Fucose Arabinos Xylose Mannose Galactose Glucose Rhodobacter Capsule Succinic acid 16.8
±0.010 16.9
±0.009 - - 21.8
±0.010 22.7
±0.000 21.8
±0.009 Malsan 16.8
±0.003 16.9
±0.009 - - 21.8
±0.001 22.7
±0.000 21.9
±0.012 Rhodobacter Blasticus Succinic acid 14.0
±0.001 14.1
±0.003 16.5
±0.004 - 18.2
±0.004 18.9
±0.002 18.2
±0.002 Malsan 20.2
±0.004 - - - 26.2
±0.005 27.3
±0.009 26.3
±0.018

As shown in Table 2, Rhodobacter capsule Latus The neutrophils that make up the glycocoli membrane of the strain consist of rhamnose, fucose, mannose, galactose, and glucose, and Rhodobacter blasticus. It was found that the strain was composed of rhamnose, fucose, arabinose, mannose, galactose and glucose.

In both strains, the glyco sugars were composed mainly of mannose, galactose, and glucose, and it was found that galactose had the most.

Rhodobacter Capsule The strain did not differ significantly in compositional sugar depending on the carbon source, but it was confirmed that in the case of the Rhodobacter blasticus strain, the glycocox generated using succinic acid as a carbon source is composed of more various types of monosaccharides.

(3) Glyco calix molecular weight measurement method

The conditions for measuring the molecular weight of Glico Calix are specifically as follows.

Plulan (shodex standard P-82) was used as a standard for measuring the molecular weight of Glico Calix, and a standard (P-200 / P-20 / P-5) was used to prevent the peaks from overlapping. ), (P-400 / P50 / Galactose (Gal)) and (P-800 / P-100 / P-10) were mixed with each 100 ul and used after filtering with a 0.45um filter.

As the HPLC column for separating the analysis sample, GS-250, GS-320, and GS-220 were used in connection, and an analytical solvent of 50 mM ammonium formate buffer (pH 5.5) was used. In addition, degassed tertiary distilled water was used as the washing solvent, and 20% ethanol was used as the column filling solvent.

The measurement method was measured using High Performance Liquid Chromatography (HPLC) connected with a Refractive Index (RI detector).

The analysis results of the molecular weight measurement chromatography of Glyco Calyx according to the above method are shown in FIG. 3, (a) cS, (b) cM, (c) bS, and (d) bM analysis results. Shows.

As a result of analyzing the same amount of glyco calix according to the above analysis method, the molecular weight of the glyco calix of the Rhodobacter encapsulation strain was measured to be 348.3 kDa when succinic acid was used as a carbon source in the basic 27S medium, and rodobacter When succinic acid was used as the carbon source for the Blasticobacterium strain, it was measured to be 687.9 kDa.

Therefore, when compared to the same amount of the Rhodobacter blasticus strain, the Rhodobacter capsuleus strain produces more moles than the Rhodobacter blastus strain by generating a small molecular weight glycocoli, It is analyzed that it forms a glycos calix.

3. Analysis of the formation amount of glycocoli according to the carbon source of the provided strain

The production amount of the glyco calix produced in the process of 1. is shown in Table 3 below.

Strain name Carbon source Glico Calix (g/L) Rhodobacter Capsule Succinic acid 2.57 Malsan 9.54 Rhodobacter Blasticus Succinic acid 1.03 Malsan 3.74

As shown in Table 3, in the case of Rhodobacter capsulotus strain, when succinic acid and malic acid were used as carbon sources, it was found that 2.57 g/L and 9.54 g/L of glycocox were generated, respectively. In the case of the Rhodobacter blasticus strain, it was found that when succinic acid and malic acid were used as carbon sources, 1.03 g/L and 3.74 g/L of glycocoli were generated, respectively.

Glico calix production was the most excellent condition was found to be cultured with a carbon source as malic acid in the Rhodobacter capsulatus strain.

Therefore, it was found that the production amount of glyco calix of Rhodobacter capsulatus strain using malic acid as the main carbon source of the culture medium was 9.54 g/L, indicating a significantly high production of glyco calix.

Although the embodiments of the present invention have been described in detail above, the scope of rights of the present invention is not limited to this, and various modifications and variations are possible without departing from the technical spirit of the present invention as set forth in the claims. It will be obvious to those of ordinary skill in the field.

Claims (4)

Method of culturing Rhodobacter capsulatus strain, Rhodobacter blasticus strain or a mixed strain thereof using a medium using malic acid as a carbon source.
The method of claim 1, wherein the medium further comprises an iron citrate solution, KH ₂ PO ₄ , MgSO ₄ , NaCl, NH ₄ Cl and CaCl ₂ .
The method of claim 1, wherein the medium comprises the carbon source in an amount of 0.01 to 0.1 wt% based on the weight of the entire medium.
According to claim 1, The culture is pH 6.5 to 7, 0.5 to 5 (v/v)% strain based on the total medium volume, inoculated into the medium, is carried out at 25 to 37° C. under aerobic anaerobic conditions. Way.

Images