KR102050338B1 - Mass culture method for antimicrobial strain - Google Patents

Abstract

The present invention provides a method for mass culturing of antimicrobial active strains capable of reliably and effectively culturing and producing highly functional active substances using marine-derived microorganisms and microbial metabolites.

Description

Mass culture method for antimicrobial strain

The present invention relates to a method for mass culturing strains, and more particularly, to a method for mass culturing antimicrobial active strains.

Until now, research on natural materials with physiological activity from natural materials has been mainly conducted on land resources, but economic trends are declining due to the depletion of new sources through many years of research. The developed countries are intensively researching natural products derived from marine microorganisms. As a result, about 5500 new substances have been developed in marine organisms. They are being developed competitively as medicines, health supplements, cosmetics, etc., and about 40 substances have been patented for pharmaceutical use, and five of them are being developed as products. Currently oceanologists are keen on finding new marine microbes and developing microbes that produce natural materials with new structures from the ocean. The development of natural materials for marine-derived microorganisms is global, and recently, global pharmaceutical companies are moving to chemical-based antibiotics and strengthening their strategies to enter bio-derived materials and to enter the antimicrobial market using microorganisms or their metabolites. In particular, the development of the next generation of marine microorganism-based antimicrobial active substances and feed additives containing them will be an opportunity to secure leading technologies in the fisheries technology field by joining the core trend of the current government policy to lay the foundation for national development through creative technology. see.

On the other hand, due to climate change caused by global warming, fish diseases are increasing significantly. Fish disease occurs when the balance of environment, pathogens, and anti-medical forces is broken, and the mortality rate from disease is rapidly increasing. For example, in the case of flounder, the damage caused by bacterial diseases such as Vibrio anguillarum, Vibrio harvevi, Edwardsiella tarda, Streptococcus iniae, etc. is increasing. The total amount is 48 million and the mortality rate is close to 40%. In the field survey of the flounder farms in Jeju, economic damage was estimated at 10 billion won annually based on 2009 flounder production, total bacterial infection rate and mortality rate. Damage is occurring. Therefore, the need for alternative antibiotics, rather than antibiotics, to promote the prevention and growth of bacterial diseases of flounder. Although antibiotics have advantages such as ease of use and effectiveness of treatment. Problems such as the generation of resistant bacteria and the retention of antibacterial substances in the body. Therefore, much research has been focused on the development of probiotics to suppress diseases occurring in aquaculture fish and to maintain the health of fish. In particular, we have been searching for non-pathogenic bacteria that produce antimicrobial, antifungal and antiviral substances from Bacillus sp. Such as Lactobacillus plantarum, Bacillus coagulans and Bacillus subtilis . The product is being developed steadily, but it has not received much attention in the market due to the lack of antimicrobial effect. Therefore, it is urgent to develop a formulation using antimicrobial materials using new marine-derived microorganisms and microbial metabolites. It is not replacing. In this regard, Republic of Korea Patent No. 0230527 discloses Sporolactobacilli K9 isolated from Kimchi, a mass culture method of the bacteria and products using the same.

However, in the case of the prior art, it does not provide an optimal medium composition for mass cultivation of Bacillus strains, but is not particularly suitable for mass culturing processes for industrial use.

The present invention has been made to solve various problems including the above problems, and an object of the present invention is to provide an economical and efficient mass cultivation method of marine microbial antimicrobial active strains. However, these problems are exemplary, and the scope of the present invention is not limited thereby.

According to an aspect of the present invention, after inoculating the antibacterial Bacillus strain in a culture medium containing 1.2 to 2.8% by weight of carbon source and 2.0 to 3.8% by weight of nitrogen source temperature 28 ~ 32 ℃, pH 6.5 ~ 7.5, air injection amount 0.3 ~ 0.7 vvm, and characterized in that the incubation for 50 to 96 hours at the culturing conditions of 50 to 150 rpm, the antimicrobial activity enhanced mass culture method of the antibacterial Bacillus strain is provided.

According to one embodiment of the present invention made as described above, due to the reduction in production cost, economical and antimicrobial material production can also implement a mass culture effect of stable antimicrobial active strains. Of course, the scope of the present invention is not limited by these effects.

1 is a plate medium and ampoule photograph of marine microorganism Bacillus sp .
Figure 2 is a graph analyzing the bacterium density (bar graph) and antimicrobial capacity (line graph) of Bacillus sp .
3 is a graph analyzing the bacterium density (bar graph) and antimicrobial activity (line graph) of Bacillus sp. Strain according to the type of nitrogen source.
Figure 4 is a graph analyzing the bacterial density (bar graph) and antimicrobial capacity (line graph) of Bacillus sp. ( Bacillus sp. ) Strain according to the content of the carbon source.
5 is a graph analyzing the bacterium density (bar graph) and antimicrobial capacity (line graph) of Bacillus sp .
Figure 6 is a photograph showing the appearance of 30L Jar fermentor (fermenter) used to establish the conditions of mass culture of Bacillus sp .
7 is a graph analyzing the bacterium density (bar graph) and antimicrobial activity (line graph) of Bacillus sp .
8 is a graph analyzing the bacterial density (bar graph) and antimicrobial activity (line graph) of Bacillus sp .
9 is a graph analyzing the bacterium density (bar graph) and antimicrobial activity (line graph) of Bacillus sp .
10 is a graph analyzing the bacterium density (bar graph) and antibacterial activity (line graph) of Bacillus sp .
11 is a graph analyzing the production amount of crude surfactin according to the carbon source content.
12 is a graph analyzing the yield of crude bacillomycin according to the amount of dissolved oxygen in the medium.
Figure 13 is a graph analyzing the content of crude surfactin produced in a pilot plant of 20KL.
Figure 14 is a graph analyzing the mass production of the Bacillus sp. ( Bacillus sp. ) Strain according to the amount of dissolved oxygen.
15 is a graph analyzing the production amount of Crude surfactin according to the addition of Fe ⁺² ion.
Figure 16 is a graph analyzing the production of the bacterial density and the antimicrobial material according to the addition of Fe ⁺² ions.
Figure 17 is a schematic diagram of the mass production process and raw material production system of marine microorganism Bacillus sp .

Definition of Terms:

As used herein, the term "probiotics" refers to living organisms that reach the intestine to produce lactic acid and to produce acidic bacteria that can not grow, harmful bacteria can grow. It has long been used in the food industry because it can be converted to other carbohydrates and sugars by lactic acid. Recently, research is actively conducted on feed additives or antimicrobial agents that can solve problems such as the occurrence of resistant bacteria of antibiotics used in large quantities to prevent disease of fish in fish farms and the retention of antimicrobial substances in the body.

As used herein, the term "pilot plant" refers to a small plant that is being constructed experimentally before a new process or new product is introduced. It is a small test facility constructed in preparation for the construction of).

As used herein, the term "bacillomycin" is an antifungal substance isolated by acidic precipitation and ethanol extraction from Bacillus subtilis subtilis culture. It is a colorless microcrystal with a molecular weight of about 1,000 and is a weakly acidic, nonribosomal cyclic polypeptide. . Basillomycin has the structure of Formula 1:

(Formula 1)

As used herein, the term "surfactin" is a very powerful surfactant commonly used as an antibiotic and is an antifungal substance produced by the bacterial cyclic lipopeptide and the Gram-positive bacterium Bacillus subtilis . The surfactants have been shown to exhibit effective properties such as antimicrobial, antiviral, antifungal, antimycoplasma and hemolytic activity.

Detailed description of the invention:

According to an aspect of the present invention, after inoculating the antibacterial Bacillus strain in a culture medium containing 1.2 to 2.8% by weight of carbon source and 2.0 to 3.8% by weight of nitrogen source temperature 28 ~ 32 ℃, pH 6.5 ~ 7.5, air injection amount 0.3 ~ 0.7 vvm, and characterized in that the incubation for 50 to 96 hours at the culturing conditions of 50 to 150 rpm, the antimicrobial activity enhanced mass culture method of the antibacterial Bacillus strain is provided.

In the mass culture method, the antibacterial Bacillus strain may be Bacillus ( Bacillus sp.) SW 1-1 strain (KCCM 11517P) or Bacillus ( Bacillus sp.) 2-4 strain (KCCM 11107P).

In the mass culture method, the culture medium is 1 to 2 parts by weight of glucose, 0.1 to 1 parts by weight of casein hydrolyzate, 0.1 to 1 parts by weight of yeast extract, 2 to 3 parts by weight of soybean meal, potassium diphosphate 0.05 to 0.5, 0.1 to 2 parts by weight of sodium chloride, 0.05 to 0.5 parts by weight of ammonium sulfate, 0.05 to 0.5 parts by weight of calcium chloride, 0.01 to 0.1 parts by weight of magnesium sulfate, and 0.0005 to 0.005 parts by weight of manganese sulfate.

In the mass culture method, the nitrogen source may be a yeast extract (yeast extract) and the culture medium may further include 3 to 5 mM Fe ²⁺ ions and the dissolved oxygen content to be maintained at 15 to 25% Can be.

The production of aquaculture flounder produced in Jeju is more than 20,000 tons in Korea and about 10,000 tons of aquaculture flounder produced mainly in Wando, Jeolla-do. The flounder is clean and stable in the land-based aquaculture. The groundwater, which maintains the water temperature, is capable of producing and raising large quantities of seedlings throughout the year regardless of the season. However, with the increase of farms and long-term farming, the disease is also increasing. In particular, bacterial diseases such as Vibrio anguillarum, Vibrio harvevi, Edwardsiella tarda, Streptococcus iniae, etc., are increasing recently , and they appear as a major risk factor for improving the productivity of flounder because it is widespread and causes massive mortality from flounder fry. have. Recently, flounders infected with bacterial diseases occur throughout the year, and the damage is increasing year by year. However, until now, there are no effective control measures, and vaccines have recently been developed. In terms of economics, the frequency of use is not very high.

In Korea, a large number of antibiotics are used to treat bacterial diseases, of which oxytetracycline accounts for 74%. However, despite the long period of drug use and proper concentration due to the overuse of antibiotics, over 80% of all fish species are resistant to antibiotics, and antibiotic-resistant bacteria are released to the outside of the farm, In the offshore farms, with the occurrence of resistant bacteria, many studies have been focused on the development of eco-friendly probiotics as an alternative to antibiotics for bacterial diseases that cause diseases in farmed fish. The impact on farmed fish is not clear and the inefficiency of feeding large quantities is not enough to replace the antibiotic market yet.

The present inventors have developed the antimicrobial active strain mass culture method that can cultivate and produce stable and effective high functional active materials using marine-derived microorganisms and microbial metabolites as a result of recognizing the above problems.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited to the embodiments disclosed below, but can be implemented in various forms, and the following embodiments are intended to complete the disclosure of the present invention, the scope of the invention to those skilled in the art It is provided to inform you completely.

Example 1 Establishment of Industrial Medium Composition of Marine-derived Microorganisms

In order to investigate the optimum industrial culture medium for liquid culture of marine microorganism Bacillus SW 1-1 (KCCM 11517P), carbon source, nitrogen source and inorganic salts were investigated in 30L Jar fermentor based on preliminary experimental results. The media components were selected based on yield (cfu / ml) and antagonistic ability.

1-1: Storage and Cultivation of Marine-derived Microorganisms

Bacillus SW 1-1 strain (KCCM 11517P) used in the present invention was used to confirm the possibility of industrialization through mass culture according to the use of industrial medium received technology transfer through the National Fisheries Research and Development Institute. The medium for strain storage was TSA (tryptic soy agar) plate solid medium, inoculated with the bacteria on the plate solid medium and incubated at 30 ℃, 24 hours in a constant temperature incubator and used for refrigeration and TSA plate every two weeks. Subcultures were used on solid media. The spawn strain was used by sterilizing 100 ml of TSB (typtic soy broth) medium in a 500 ml Erlenmeyer flask and shaking culture at 30 ° C., 120 rpm and 6 hours. In addition, the culture medium incubated in TSB medium for 24 hours for long-term storage of the strain was mixed with a sterile skin milk as a cryopreservative 1: 1 and stored at -80 ° C (Fig. 1).

1-2: Selection of industrial medium composition

The bacterial density of the Bacillus SW 1-1 strain (KCCM 11517P) culture complete solution was determined using a spectrophotometer (660 nm) and the antibacterial activity of the culture complete solution was determined using a paper disk method. By comparison. Specifically, after incubating Edwardsiella tarda , a representative fish pathogenic microorganism, in BHI broth medium, E. tarda at a concentration of 1.0 x 10 ⁸ cfu / ml was plated on BHI agar. Subsequently, a 0.8 cm diameter paper disk was placed up, and 50 µl of each culture completed solution was added and incubated at 25 ° C. for 24 to 48 hours, and then the size of the formed transparent ring was measured and compared.

1-3: Carbon source selection

The effect of carbon source on the culturing of Bacillus sp. Strain was investigated by changing only the carbon source without changing the remaining nutrients in the basic medium. All carbon sources (sucrose, mannitol, fructose, glucose, casein hydrolysate, glycine, cystein, CaCO3, yeast extract, tryptone) added 0.5% equal to the carbon content of the base medium, incubated for 48 hours at 30 ℃, 100 pm conditions The bacterial density and antibacterial activity were observed.

As a result, when the casein hydrolysate was treated as the carbon source, the number of viable cells was the highest, and the antimicrobial activity was the highest when the glucose was treated. Based on the results, glucose and casein hydrolysates were selected as carbon sources (FIG. 2).

1-4: Nitrogen source selection

The effect of nitrogen source on the cultivation of Bacillus SW 1-1 strain (KCCM 11517P) was investigated by changing only the nitrogen source without changing the remaining nutrients in the basic medium. As with the carbon source selection experiments, all nitrogen sources were added with 0.5% of the same as the basic medium to observe the bacterial density and antimicrobial activity.

As a result, when the yeast extract (yeast extract) and soybean meal (soybean meal) was used, the highest density and antibacterial activity was found to be the nitrogen source (Fig. 3).

1-5: Determination of Carbon Source and Nitrogen Source Concentration

The bacterial density and antimicrobial activity of the selected carbon and nitrogen sources were investigated. Specifically, the carbon source (glucose hydrolyzate, soybean meal) and the carbon source (casein hydrolyzate, glucose) and the nitrogen source (yeast extract, soybean meal) are respectively fixed at a concentration of carbon source, nitrogen source, and inorganic salt in a basic medium. The concentration of the degradation product was 3: 1 and the nitrogen source was treated after mixing the concentration of yeast extract and soybean meal at 1: 5.

As a result, when the total carbon source was 2.0%, the nitrogen source was 3.0%, the concentration of the highest density was the highest, showing the strongest antibacterial ability (Figs. 4 and 5). Based on the above results, the optimal industrial medium composition of the genus Bacillus SW 1-1 strain (KCCM 11517P) was established and shown in Table 1 below.

Furtherance content Glucose 1.5 Casein hydrolysate 0.5 Yeast extract 0.5 Soy bean meal 2.5 K ₂ HPO ₄ 0.1 NaCl 0.4 (NH ₄ ) ₂ SO ₄ 0.1 CaCl ₂ ㅇ 2H ₂ O 0.1 MgSO ₄ ㅇ 7H ₂ O 0.05 MgSO ₄ ㅇ 5H ₂ O 0.001

Example 2: Establish Production Conditions for Mass Culture (2KL)

Based on the industrial medium composition, the culture conditions on a 30L Jar fermentor (fermenter) were investigated in order to establish the conditions of pilot culture of marine microorganism Bacillus SW 1-1 strain (KCCM 11517P). First, the production conditions of mass culture of 2KL scale were established using the culture engineering method based on the culture condition of 30L scale, and the basic culture conditions were the culture conditions of the general Bacillus SW 1-1 strain (KCCM 11517P). (30 ° C., 100 rpm, 0.5 vvm, 0.5 kgf / cm ² ) was used (FIG. 6).

2-1: Analysis of culture efficiency according to temperature

Using the established industrial medium composition, the optimum temperature for mass culture of Bacillus strains was identified. Specifically, inoculated with 0.5% Bacillus SW 1-1 strain (KCCM 11517P) to 30L Jar fermenter containing sterilized 15L medium and incubated at 20 ℃, 25 ℃, 30 ℃, 35 ℃ and 40 ℃ temperature conditions. .

As a result, the bacterial density was highest when incubated at 30 ℃, 35 ℃ but the antimicrobial activity was the highest at 30 ℃, so the culture temperature was selected to 30 ℃ (Fig. 7).

2-2: Culture efficiency analysis according to pH

Since the initial pH of the industrial medium is an important factor in determining the initial cultivation rate of the seed medium in the industrial medium, the initial pH before starting the seed inoculation using 20% NaOH after sterilizing the industrial medium to select the optimal pH for the culture is pH 5 ~ pH. It was adjusted to 9 and analyzed for the bacterial density and antimicrobial activity after 48 hours of culture.

As a result, it was shown that the Bacillus SW 1-1 strain (KCCM 11517P) has excellent bacterial density and antimicrobial ability between pH 6 and 8, and showed the highest antimicrobial activity, especially at pH 7 (FIG. 8).

2-3: Analysis of culture efficiency according to air injection amount

In order to confirm the optimum air injection amount during the culture, the air injection amount was adjusted to 0.1 to 2.0 vvm, and after 48 hours of incubation, the bacterial density and antimicrobial activity were analyzed.

As a result, the amount of air injection increased to 1.5 vvm as the amount of air injection increased, but the antimicrobial ability was most found at 0.5 vvm, and the amount of air injection decreased as the amount of air injection increased (FIG. 9).

2-4: culture efficiency according to the stirring speed

In order to observe the effect of bacterial density and antimicrobial activity of Bacillus SW 1-1 strain (KCCM 11517P) according to the agitation speed, the agitation speed was adjusted to 50, 100, 150, 200, and 250 rpm, respectively, and then cultured for 48 hours. The bacterial density and antimicrobial activity were investigated.

As a result, the agitation density was increased as the stirring speed was increased, but as the stirring speed was increased based on 100 rpm, the antimicrobial activity was found to decrease (FIG. 10).

2-5: Establishment of pilot plant production process

The production process of the pilot plant (2 KL) of the genus Bacillus SW 1-1 strain (KCCM 11517P) was established based on the industrial medium composition and the optimum culture conditions established above. First, the scale-up step was carried out in the steps of Bacillus ampoules-> 50 ml-> 10 L-> 2 KL and each dose was set to 0.5%. The culture conditions and incubation time for each step are shown in Table 2 below.

Process Culture volume Culture condition Inoculation amount Incubation time (hr.) A unit ample -80 ℃ B unit 50 ml 30 ℃, 120 rpm 1 ampoule 8 C unit 10 L 30 ° C, 120 rpm 50 ml 8 Main 2 KL 30 ℃, 100 rpm 10 L 48

2-6: Pilot Plant Production Comparison

After establishing the production process of the pilot plant (2 KL) through this, the culture and production proceeded. Specifically, B unit and C unit used TSB medium in the Erlenmeyer flask, and the seed work was performed in a sterile chamber to prevent invasion of external contaminants, and the seed culture was carried out using a shaker incubator. Main culture was prepared by sterilizing 2 KL of industrial medium in 4 KL Jar-fermentor, and inoculated when C unit incubation had elapsed for 8 hours. Inoculation was carried out with flame inoculation to prevent invasion of external contaminants, sampling was carried out every 1 hour during the cultivation, analysis of substances showing antimicrobial substances (crude surfactin, crude bacillomycin), changes in carbon source content, and dissolved in medium. The amount of oxygen was measured.

As a result, the change of carbon source and dissolved oxygen in the medium appeared rapidly after 6 hours of cultivation. Among the two antimicrobial substances, crude bacillomycin was cultured for about 8 hours and crude surfactin was cultured for 11 hours. Since it started to occur after 16 hours of culture did not appear to change the content of the antimicrobial material (Figs. 11 and 12).

Example 3: Establishment of stable mass culture (20 KL) technology of antimicrobial material

Based on the pilot plant of the 2 KL scale applied in the above embodiment was applied to the mass production process system of 20 KL for the reduction of production cost and increase of the active material.

3-1: Confirmation of Substance Production of Bacillus Strains

Based on the mass production process system of the 2 KL scale, material production of the Bacillus strain was observed at the 20 KL scale.

As a result, the total incubation time was increased (48h.-> 80h.) Compared with the 2 KL pilot plant, and the content of the antimicrobial josufectin was decreased (9.2 g / L-> 7.8 g / L) (Fig. 13).

3-2: Confirmation of material production by controlling dissolved oxygen

In general, oxygen plays a major role in the production of secondary metabolites such as metabolism, growth of strains and antibacterial substances in the culture of strains. As the bacterial density of Bacillus SW 1-1 strain (KCCM 11517P) increases, The amount of dissolved oxygen decreased, and the amount of dissolved oxygen in the log growth and growth stages of most bacteria was less than 5%. Based on this, dissolved oxygen was continuously maintained at 20% or more in culture, and bacterial growth and antimicrobial production were compared. First, in order to maintain the dissolved oxygen amount, the oxygen input amount was increased from 0.5 vvm-> 1.5 vvm and the stirring speed was increased stepwise from 100 rpm-> 170 rpm to maintain the dissolved oxygen content in the medium at 20% or more. Oxygen dose and stirring speed were adjusted stepwise to 0.5 vvm, 100 rpm.

As a result, after 24 to 32 hours of cultivation, the contents of josectectin and joseobacillomycin were the highest, and as the culture continued, it was observed that the glucose content rapidly decreased (FIG. 14).

3-3: Fe ²⁺  Material production and uniformity confirmation by adding ion

Since the major antimicrobial material produced during the culture of marine microorganisms in the culture of the peptide is formed peptide structure, Fe ²⁺ ions were added to induce stable peptide formation, and then the contents of the antimicrobial substance and the bacterial density were investigated.

As a result, when the addition of 4 mM Fe ²⁺ ions compared to the control it was observed that the content of the crude coughfectin is about 20 mg / L per hour and also showed the highest bacterial density (Figs. 15 and 16).

3-4: Mass Cultivation Process

Through the industrial medium composition and mass culture process system (20 KL), the bacterium density and antimicrobial production amount of marine microorganism Bacillus SW 1-1 strain (KCCM 11517P) was established (FIG. 17). In addition, the culture-specific and post-culture process of the mass culture process system (20 KL) of the marine-derived microorganism Bacillus SW 1-1 strain (KCCM 11517P) of the present invention is shown in Table 3 below.

Main culture Temp .: 30 ℃ RPM: 100 rpm (control 170 rpm, DO 20% or more) Air: 0.5 vvm (control 1.5 vvm, DO 20% or more) pH: Initial pH 7.0 Working volume: 50% Inner pressure: 0.5 kgf / cm ² Incubation time: 72 h. Spray drying Centrifugation: 5 times concentrated Excipients: Toal 20% (Corn starch 10%, Oxidized starch 7%, MSG 3%)

In conclusion, the mass culturing method of the Bacillus SW 1-1 strain (KCCM 11517P) having the antimicrobial activity of the present invention can stably and effectively cultivate and produce highly functional active substances using marine microorganisms and microbial metabolites. It can be used to produce environmentally friendly feed additives for the prevention of bacterial diseases of fish diseases, especially the flounder, an economical fishery in the fisheries industry.

On the other hand, the inventors of the present invention Bacillus sp. ( Bacillus sp.) 2-4 strain (KCCM 11107P) also significantly increased the content of the antimicrobial substances such as bacillomycin and suctin described above when using the above-described culture method It was confirmed. Therefore, it can be seen that the method of the present invention is not only specifically applied to the genus Bacillus SW 1-1 strain (KCCM 11517P) but is a general-purpose technology applicable to various antibacterial Bacillus strains.

Although the present invention has been described with reference to the above-described embodiments, these are merely exemplary and will be understood by those skilled in the art that various modifications and equivalent other embodiments are possible. Therefore, the true technical protection scope of the present invention will be defined by the technical spirit of the appended claims.

Depositary Name: Korea Microorganism Conservation Center (overseas)

Accession number: KCCM11517P

Trust Date: 20140206

Depositary Name: Korea Microorganism Conservation Center (overseas)

Accession number: KCCM11107P

Deposit Date: 20101006

Claims (6)

Bacillus SW 1-1 strain (KCCM 11517P) was inoculated into a culture medium containing 1.2 to 2.8 wt% of a carbon source and 2.0 to 3.8 wt% of a nitrogen source, followed by a temperature of 28 to 32 ° C., a pH of 6.5 to 7.5, and an air injection amount of 0.3 to Culture method for enhancing the antimicrobial activity of the Bacillus genus SW 1-1 strain (KCCM 11517P), characterized in that incubated for 0.7 to vvm, and 50 to 96 hours at the cultivation conditions of a stirring rate of 50 ~ 150 rpm. delete The method of claim 1,
The culture medium is 1 to 2 parts by weight of glucose, 0.1 to 1 parts by weight of casein hydrolyzate, 0.1 to 1 parts by weight of yeast extract, 2 to 3 parts by weight of soybean meal, 0.05 to 0.5 of potassium diphosphate, 0.1 to 2 parts by weight of sodium chloride, A culture method comprising 0.05 to 0.5 parts by weight of ammonium sulfate, 0.05 to 0.5 parts by weight of calcium chloride, 0.01 to 0.1 parts by weight of magnesium sulfate and 0.0005 to 0.005 parts by weight of manganese sulfate. The method of claim 3,
Nitrogen source is a yeast extract, a culture method. The method of claim 3,
The culture medium further comprises 3 ~ 5 mM Fe ²⁺ ions, culture method. The method of claim 1,
Dissolved oxygen is maintained at 15 to 25% culture.

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