KR20020096020A - A method for producing arachidonic acid by fed batch culture of a Mortierella sp. strain - Google Patents

Abstract

PURPOSE: Provided is a method for mass-producing an arachidonic acid with inexpensive costs by generating a culture condition, in which mortierella sp. strain can produce a large quantity of arachidonic acid. CONSTITUTION: In the method for culturing mortierella sp. strain, glucose and corn steep powder are used as a carbon source and nitrogen source, respectively; 7-40% of ammonia solution is added to a fermenter so as to adjust a pH, whenever pH is dropped below the specific point in the range of 5-6 during cultivation; and the ammonia solution added to the fermenter is also used as a nitrogen source. Particularly, the specific point is pH 5.5. The cultivation is initiated by using an initiation medium containing 20g/L or more of glucose and 10g/L or more of corn steep powder.

Description

A method for producing arachidonic acid by fed batch culture of a Mortierella sp. strain}

The present invention relates to a method for producing arachidonic acid in large quantities using a strain of the genus Mortierella with high lipid content. More specifically, the present invention relates to a method for mass production of arachidonic acid through oil culture.

Arachidonic acid is a polyunsaturated fatty acid (5,8,11,14), which is also represented by -6 or 20: 4 carbon atoms in the -6 series having four cis double bonds at the 5,8,11,14-positions. A type of 8,11,14 eicosatequienoic acid, linoleic acid or alpha-linolenic acid, which is an essential fatty acid that is not produced in mammals and is consumed mainly through plant foods. acid) is produced through the synthesis route of gamma-linolenic acid and dihomo-linolenic acid and is an important component of cell membrane. In addition, arachidonic acid is a precursor of prostaglandins, leucotrienes, and thromxane, which are produced through various pathways of the arachidonic acid cascade, and these eicosanoids are developed and blooded. It is a bioactive substance with important functions involved in regulation of the immune system, inhibition of platelet aggregation, inhibition of plasma cholesterol, skin protection and the like.

Arachidonic acid is synthesized in the body in the amount needed by healthy adults, but in infants and the elderly, the enzyme activity of delta5-desaturase and delta6-desaturase is included in the synthesis pathway. Because of this low production of arachidonic acid is low, so a dietary supply of arachidonic acid is required.

Arachidonic acid (ARA) is an omega-6 class of long-chain polyunsaturated fatty acids (PUFA, 20: 4), which is not synthesized directly in mammals, but instead contains linoleic acid, an essential fatty acid ingested from plant foods. It is an important in vivo fatty acid synthesized as a precursor. Arachidonic acid is a major component of cell membranes in vivo and is also used as a precursor for the synthesis of various ecosanoids such as prostaglandin, thromboxane, and leucotriene, which are involved in metabolic regulation. do. These eicosanoids exhibit modulatory effects on fat protein metabolism, blood flow, blood vessel color and platelet activation. However, the elderly, the sick and the infants need a diet of arachidonic acid because the production of arachidonic acid is lowered in the body (Burre et al., Lipids, 25: 354-356 (1990)). In addition, omega-3 fatty acid content in the blood cells was decreased, scaly dermatitis was observed, and symptoms such as perception, muscle strength, difficulty walking, lower extremity pain, and visual impairment were reported. Is contained in about 0.3% (Suzuki et al., 1988), and therefore, the addition of arachidonic acid is required to further breast-feed infant formula containing no arachidonic acid. Mass production of arachidonic acid is needed for the baby food and dietary supplement markets.

Clandin et al. Proposed a blend of egg yolk, fish oil or erythrocyte phospholipids and vegetable oils as a fat component of baby food in US Pat. No. 4,670,285. However, fish oil contains eicosapentaenoic acid (EPA) and EPA is known to inhibit the synthesis of arachidonic acid in infants (Carson et. Al., INFORM, 1: 306, 1990). In addition, yolk contains relatively low concentrations of arachidonic acid, so mixtures of yolk or fish oil are not suitable for baby food. Arachidonic acid is an animal oil and there are many difficulties for its commercial production. The results reported so far for the commercial production of arachidonic acid are as follows.

In 1987, Das et al. Flask culture using malt extract medium using M. isabellina 214 produced 505 mg / L of arachidonic acid, Shimizu et al. Suntory, Kyoto University. Co., Ltd. was isolated from Mortierella elongata IS-5, a strain containing 10% glucose, 0.5% polypeptone and 0.3% yeast extract, in culture medium at pH 6.0 and 24 ° C for 4 days. Incubated in the flask to produce 0.99 mg / mL (22 mg / g dry cells). Also in 1988 M. Alpina ( M. alpina) was fed a fed-batch culture at 20 ° C. using a medium containing glucose and yeast extract, and the yield of mycelia was increased by changing the incubation temperature to 12 ° C. at the beginning of growth. ) And shortened the incubation time. And M. , who was separated in 1989 . When Alpina IS-4 was incubated in a 5L bench-scale fermentor using a medium containing glucose and yeast extract, 3.6 g / L of arachidonic acid was produced and the cells continued. In aging, the content of arachidonic acid increased to 67.4% of total fatty acids. In 1988, Totani et al., Lion. Alpina ( M. alpina) IFO 8568 was incubated for 20 days at 20 ° C. in a medium containing potato paste and glucose to produce 11.8 g / kg- medium of arachidonic acid, where the content of arachidonic acid was 68% of the total lipid content. It was. In 1990, Certik et al., Czechoslovakia, produced 0.96 g / L (12.7 mg / g dry cells) arachidonic acid when flasks were cultured using Czapek liquid medium containing glucose and yeast extracts. It was. In 1991, Bajpai et al. Alpina ( M. alpina) ATCC 32222 was incubated for 6 days under optimal culture conditions to produce 3.7 g / L of arachidonic acid. In 1994, Chang et al. Alpina ( M. alpina) CBS 210.32 was incubated for 5-7 days to produce 0.86-0.96 g / L arachidonic acid. They also isolated a fungus named Wuji-H4 from the soil in 1997 and at 24 ° C for 5 days in an optimized medium of 99.7 g / L soluble starch, 12.6 g / L yeast extract, 3 g / L KH ₂ PO ₄ . Incubation produced 3.9 g / L of arachidonic acid. In 1994, Certik, Czechoslovakia, was isolated from soil. Alpina (M.alpina) S-17 3% glucose by using a semi-synthetic (semisynthetic) medium containing by 14 days in culture flask at 28 ℃ was producing lipid of 2.1 g / L, wherein the content of arachidonic acid is the total 50% of the lipid content. In 1995, Li et al. Alpina ( M. alpina) VW-1 was incubated in shake flasks and fermenters for 7 days to produce 5.3 g / L of arachidonic acid. However, because the medium used canola oil (canola oil) and insoluble soy flour (insoluble soy flour), there was a problem in the recovery of arachidonic acid.

Recently, a health-oriented food market related to polyunsaturated fatty acids has been formed in Korea, and its market size is expected to soar, and imports from overseas are expected to surge. Such arachidonic acid can be produced by extraction from pig liver, sardine and egg yolk, but the content is very low as 5% or less, and recently, research for mass production of arachidonic acid from microorganisms has been actively conducted. However, the reality is that the technology for mass production of arachidonic acid has not been developed properly.

The present invention is to solve the above problems, an object of the present invention is to provide a method for the mass production of arachidonic acid at low cost by forming a culture condition that can produce a large amount of arachidonic acid strains of the genus Mortierella It is. Another object of the present invention is to provide a fed-batch culture method capable of cultivating a mycelia of small pellets containing arachidonic acid and lipids at a high concentration.

1 is a graph showing the monitoring of the change according to the culture time in the culture method of Example 1 of the present invention.

Figure 2 is a graph showing the monitoring the change according to the incubation time in the culture method of Example 2 of the present invention.

Figure 3 is a graph showing the monitoring of the change according to the incubation time in the culture method of Example 3 of the present invention.

Figure 4 is a graph showing the monitoring the change according to the incubation time in the culture method of Example 4 of the present invention.

Figure 5 is a graph showing the monitoring of the change according to the incubation time in the culture method of Example 5 of the present invention.

Figure 6 is a graph showing the monitoring of the change according to the incubation time in the culture method of Example 6 of the present invention.

Figure 7 is a graph showing the monitoring the change according to the incubation time in the culture method of Example 7 of the present invention.

In order to achieve the above object, the production method of arachidonic acid through the fermentation of the strain of the genus Mortierella, according to the present invention, in the culture of Mortierella sp. Strain, using glucose as a carbon source And using cornstalk powder as a nitrogen source, and when the pH falls below a specific pH within the range of pH 5 to 6, 7 to 40% of ammonia solution is supplied to the fermenter to adjust the pH, and The ammonia solution is also used as a nitrogen source.

In the production method of arachidonic acid through the oil-feeding culture of the genus Mortierella according to the present invention, the specific pH is characterized in that 5.5.

In the production method of arachidonic acid through the fermentation culture of the strain of the genus Mortierella according to the present invention, the specific pH is 5.5 up to a specific time in the 68 to 76 hours after culture, 6.0 after the specific time It is characterized by.

In the production method of arachidonic acid through the oil-feeding culture of the genus Mortierella strain according to the present invention, the culture is an initial medium containing more than 20g / L glucose as a carbon source and 10g / L cornstalk powder as a nitrogen source It is characterized by starting with.

In the production method of arachidonic acid through the fed-batch culture of the genus Mortierella strain according to the present invention, the culture is in the middle of the culture, when the concentration of the initially supplied glucose is less than 15g / L fermentation of additional glucose It characterized in that the supply to.

In the production method of arachidonic acid through the fermentation culture of the genus Mortierella strain according to the present invention, the culture is in the middle of the culture, while adding the MnSO ₄ in the fermentor to induce the growth of the cells in pellet form arachidone in lipids It is characterized by increasing the content of acid.

In the production method of arachidonic acid through the fed-batch culture of the genus Mortierella strain according to the present invention, the culture is characterized by maintaining the amount of dissolved oxygen in the culture medium and inducing the growth of pellets in the culture medium by changing the stirring speed It is done.

In the production method of arachidonic acid through the fermentation culture of the genus Mortierella strain according to the present invention, the genus Mortierella strain is characterized in that the strain belonging to Mortierella alpina ( Mortierella alpina ).

In the production method of arachidonic acid through the fermentation culture of the genus Mortierella strain according to the present invention, the genus Mortierella strain is characterized in that the mortierella Alpina DSA-12.

Hereinafter will be described in more detail with respect to the production method of arachidonic acid through the fertilization culture of the genus Mortierella strain according to the present invention.

Production method according to the invention is characterized by using a Mortierella sp . Preferably it is a Mortierella alpina strain, and most preferably Mortierella alpina DSA-12.

Mortierella alpina DSA-12 strain can be easily obtained in nature as follows.

Using a M1 medium containing stearic acid, yeast extract, malt extract and a small amount of inorganic salts, the high production strains of arachidonic acid among the fungi isolated from nature were searched. The isolated strains were again cultured in M2 medium, a selection medium containing glucose, yeast extract, malt extract, bacto peptone and inorganic salts, to select 10-20 strains with high arachidonic acid content. The selected strains are further cultured in M3 medium containing glucose, yeast extract, malt extract, bactopeptone and inorganic salts, and the strains of the genus Mortierella, which have the highest productivity of arachidonic acid, are selected. The strains thus selected were named Mortiella spp. DSA-12 strain. Prior to performing the production method according to the present invention, a specific strain isolation and screening procedure for obtaining a Mortiella genus DSA-12 strain is as follows.

470 types of fungi isolated from nature were searched for high production strains of arachidonic acid. M1 medium was used as a medium for separating fungi in nature, and the composition of the M1 medium is shown in Table 1 below.

Stearic acid 0.5-1.0% East Extract 0.1-0.5% Malt Extract 0.1-0.5% Ammonium Sulfate 0.01-0.05% Potassium Dihydrogen Phosphate 0.01-0.05% Magnesium sulfate 0.01-0.05% A thousand 1.5-2.0% pH 6.0-7.0

A part of the prepared soil sample was suspended in sterile saline solution and plated in M1 medium, and colonies formed while incubating at 20-28 ° C. were transferred to another M1 medium, and then each isolated strain was replated in the same M1 medium. Several fungal strains were isolated purely. Thus pure strains were inoculated in M2 liquid medium and incubated at 20 ~ 28 ℃ shaking culture, the composition of the used M2 medium is shown in Table 2.

glucose 1.0-3.0% East Extract 0.2-0.5% Malt Extract 0.2-0.5% Bactopeptone 0.2-0.5% Potassium Dihydrogen Phosphate 0.01-0.05% Magnesium sulfate 0.01-0.05% pH 6.0-7.0

The cells were recovered by centrifugation from the culture medium incubated in M2 medium for 5 to 7 days, and each of the recovered cells was washed three or more times with sterile saline solution, lyophilized, and analyzed for fatty acid composition in the cells. Seventeen strains with high acid content were isolated and screened.

In order to compare the arachidonic acid production ability of the 17 strains selected as the primary as described above was subjected to liquid culture of the strains, the composition of the M3 medium used at this time is shown in Table 3.

glucose 3.0-5.0% East Extract 0.2-0.5% Malt Extract 0.2-0.5% Bactopeptone 0.2-0.5% Potassium Dihydrogen Phosphate 0.01-0.05% Magnesium sulfate 0.01-0.05% Final pH 6.0-7.0

After sterilizing 100ml of M3 medium in a 500ml Erlenmeyer flask, the cells were inoculated and incubated at 150-200 rpm for 5-7 days at 20-28 ° C, and the cells were recovered by centrifugation. The recovered cells were dried and analyzed for dry cell weight, total lipid content, arachidonic acid content, and finally, a parent strain for producing arachidonic acid was selected.

Lipid total of cultured 17 strains as compared to the content, the growth of the cells, arachidonic acid content, etc. and finally arachidonic was the acid productivity screening excellent strain, after the identification of the strain, Mortierella alpina (Mortierella alpina) DSA-12 It was named.

The method of the present invention for producing arachidonic acid by culturing Mortierella alpina DSA-12 obtained as described above is carried out through the following process. Solid medium was used by adding 1.5% agar to GY medium (2% glucose, 1% yeast extract). The liquid medium used was glucose, corn steep powder (CSP) and ammonia, respectively. Mycelium from the agar plate was collected in a loop, placed in 5 mL of sterile distilled water, strongly vortexed, and centrifuged at 4,000 rpm for 5 minutes. The supernatant was inoculated into a 500 mL baffled flask containing 100 mL of GY medium and incubated for 2 days with stirring at 150 rpm in a shaking incubator at 25 ° C.

Hereinafter, the production method of arachidonic acid through the oil value culture of the genus Mortierella strain according to the present invention will be described in more detail.

Example 1 Fed-Batch Culture I

In order to produce arachidonic acid at a high concentration, it is necessary to produce a high concentration of bacteria first.Because of the cultivation characteristics of the fungus, the viscosity increases as the culture proceeds, and the growth of the cells is inhibited due to agitation and restriction of oxygen transfer. The content is also lowered. Therefore, in order to obtain a high concentration of the cell mass, the culture was started with 20 g / L glucose and 10 g / L CSP which is determined that the growth of the cells is not inhibited. Using a 19 L fermentor, the initial working volume was 8 L, the incubation temperature was maintained at 25 ° C., the aeration rate was 0.5 vvm, and the stirring speed was started at 150 to 200 rpm. In addition, in order to control the foam (foam) generated during the cultivation was adjusted with neorin (Neorin) or by controlling the stirring rate to control the foam generated. In addition, the substrate was supplied at this time because the glucose concentration in the medium was almost depleted when the pH was increased to 5.5 or more. A nitrogen source was supplied into the culture and at the same time, 7-14% ammonia was used to control the pH, which was automatically injected when the pH was below 5.5. The fed-batch cultivation was started with the initial operation volume of 8.0 L using glucose 20 g / L and CSP 10 g / L while adjusting the C / N ratio during the batch culture to about 20: 1. In addition, 14% ammonia solution was prepared by diluting the ammonia stock solution (28%) to maintain the pH at 5.5 during the cultivation to adjust the pH during the cultivation. 800 mL of a 800 g / L glucose solution was supplied in four portions of 200 mL (160 g glucose) at intervals of 24 hours from 48 hours to incubate the final glucose concentration to 100 g / L. At the end of the incubation, the spent ammonia solution was 260 mL, which is equivalent to the final nitrogen concentration of about 5.4 g / L, calculated as nitrogen (N). As a result, the cell mass was obtained up to 41.4 g / L at 168 hours of cultivation, and the total lipid content in the cells was about 50%, which increased continuously with the incubation time. In particular, the growth of the cells increased almost in a straight line until 7 days of culture, indicating that the ammonia supplied by adjusting the pH with respect to the carbon source can be sufficiently used as the nitrogen source. However, the low dissolved oxygen was inferior to aeration compared to the aeration, and was incubated at 200 rpm from the beginning of the culture. However, due to excessive foaming, it was lowered to 150 rpm on the second day of the culture. In order to maintain the dissolved oxygen level after 20 days, the cells were stirred while applying shear stress to the cells and raised again at 200 rpm. The reason is that agitation is more effective in increasing the amount of dissolved oxygen than adjusting the aeration to increase the amount of dissolved oxygen to maintain the dissolved oxygen amount of 20% or more. However, since the foam still occurred, the culture was continued after fixing the stirring speed at 150 rpm from the fourth day of the culture. Therefore, dissolved oxygen decreased slowly and maintained 20-30%, but dropped to 5% after switching to 150 rpm. The total lipid content increased, similar to the increase in cell mass, which was slightly higher in batches, with 49.7% at 168 hours. In addition, the content of arachidonic acid in total lipids was 34.8%, which finally yielded 7.2 g / L. The results of monitoring the glucose concentration, pH, dissolved oxygen, dry weight, total lipids, arachidonic acid production, etc. over time of the incubation process of Example 1 are shown graphically in FIG.

Example 2 Fed-Batch Culture II

The fed-batch culture was performed from 48 hours of cultivation while performing a batch culture using 20 g / L glucose and 10 g / L CSP in a 19 L fermenter. The pH was maintained at 5.5 using a 14% ammonia solution for the entire incubation time, but the concentration of sugar was measured intermittently to adjust the pH to 6.0 or higher when it was judged that sugar was not consumed. As a result, during the incubation period, the pH was adjusted to 5.5 by 14% ammonia solution and increased to 6.0 from the 3rd day of culture when it was judged that the growth of the cells was slowed and no sugar was consumed. It was made. Substrate feeding was performed by adding 200 mL of concentrated 800 mL glucose solution (1160 g / L) four times when the concentration of glucose in the medium was almost depleted when the pH increased to 6.0 or higher. The final sugar concentration was about 150 g / L to about 30 g / L. Stirring speed was started at 150 rpm, incubation temperature was 25 ℃, aeration rate was 0.5 vvm. Experimental results (see Fig. 2), the growth of the cells was continued to obtain a maximum cell weight of 62.1 g / L in 244 hours of incubation, total lipid content of 64.4%, which can finally produce 40 g / L of total lipids there was. The content of arachidonic acid in the lipid was 30.0%, which produced 12.0 g / L of arachidonic acid. The results of monitoring the glucose concentration, pH, dissolved oxygen, dry matter, total lipids, arachidonic acid production, etc. over time during the culture process according to Example 2 are shown graphically in FIG. 2.

Example 3 Fed-Batch Culture III

The fed-batch culture was performed with the final sugar concentration used being 170 g / L. The fed-batch culture for this was carried out in the same manner as in the fed-batch culture II of Example 2. That is, fed-batch culture was performed from 48 hours of cultivation while performing batch culture using glucose 20 g / L and 10 g / L of CSP in a 19 L fermenter. The pH was maintained at 6.0 for the entire incubation time to allow more 14% ammonia solution to be added. Substrate supply was achieved when glucose concentration in the culture was almost depleted at the time when the pH increased to 6.0 or higher. The concentration of glucose in the culture was increased by adding 160 mL of concentrated 800 mL glucose solution (1,850 g / L) five times. The concentration was about 40 g / L so that the final sugar concentration was 170 g / L. Stirring speed was started at 150 rpm, the incubation temperature was 25 ℃, the aeration rate was maintained at the end of the culture at 0.5 vvm. Experimental results (FIG. 3) At 300 hours of incubation, the cell mass was able to produce 70.5 g / L, and the total lipid content was 65%, which was able to produce 40 g / L of total lipid. The morphology of the cells was maintained in the form of pellets from the beginning of the culture to the end of the culture. The results of monitoring the glucose concentration, pH, dissolved oxygen, dry weight, total lipids, arachidonic acid production, etc. over time of the incubation process of Example 3 are shown graphically in FIG. 3.

Example 4 Fed-Batch Culture IV

The fed-batch culture in a 500 L fermenter (Marubishi Co. Japan) had an initial operating volume of 300 L and a final volume of 332 L. The initial pH was sterilized to 6.0 before sterilization to 5.5, and the incubation temperature and aeration were maintained at 25 ° C. and 0.5 vvm, respectively, and the stirring speed was set at 150 to 200 rpm. Neorin) was used in 3 dilutions. The initial medium was cultured using 20 g / L glucose as the carbon source and 10 g / L CSP as the nitrogen source. The addition of glucose, which is a carbon source, was supplied at intervals of one day based on 48 hours after incubation, so that the concentration of sugar was maintained at 15-20 g / L, and 14% ammonia was used for supplying nitrogen and adjusting pH. 14% ammonia was automatically supplied when the pH in the culture tank was 5.5-6.0, and the cell mass, total lipid content and arachidonic acid content were analyzed over time. The stirring speed was 150 rpm and the pH was maintained at 5.5 during the cultivation using 14% ammonia for the supply of a nitrogen source. The glucose was 8 L (6 kg glucose) with 32 L of a glucose solution of 750 g / L from 48 hours of culture. ) Was divided into 4 times and fed at intermittent intervals every day so that the final supplied glucose was 100 g / L. Experimental results (FIG. 4) A maximum cell mass of 25.5 g / L was obtained at 144 hours of culture, and the total lipid content of the cells was 40.5%, resulting in 10.2 g / L of lipid. The content of arachidonic acid in total lipids was 30.5%, producing 3.14 g / L of arachidonic acid. The results of monitoring the glucose concentration, pH, dissolved oxygen, dry matter, total lipids, arachidonic acid production, etc. over time of the incubation process of Example 4 are shown graphically in FIG. 4.

Example 5 Fed-Batch Culture V

In 500L fermenter, incubation of the cells in the form of pellets, rather than the growth of the needles, was carried out at the initial agitation speed of 200 rpm to facilitate the supply of 14% ammonia. Batch cultivation was performed up to 48 hours using 10 g / L CSP, and fed-batch culture was performed after 48 hours. That is, glucose was supplied with 32 L of glucose solution, 750 g / L, divided into 4 times of 8 L (6 kg glucose) at intervals of 24 hours after 48 hours of culture, so that the final glucose concentration was 100 g / L. When the pH falls below 5.5, 14% ammonia was supplied, but after 72 hours of incubation, the conversion was made to 6.0. In the experimental results (Fig. 5), the cell mass was able to obtain a maximum cell weight of 32.5 g / L, the total lipid content in the cell was 45.5% to finally produce 14.8 g / L of total lipid. In addition, the content of arachidonic acid in total lipids was 35.5%, producing 5.3 g / L. The shape of the cells was formed in the form of pellets to the first 48 hours, but it was released as the culture progressed, and after 72 hours, the cells were completely released and grew. The results of monitoring the glucose concentration, pH, dissolved oxygen, dry weight, total lipids, arachidonic acid production, and the like over time during the culturing process of Example 5 are shown graphically in FIG. 5.

Example 6 Fed-Batch Culture VI: Addition of MnSO ₄ Ions (1)

Using a 500L fermenter, the stirring speed was fixed at 200 rpm, and the fed-batch culture was performed in the same manner as the fed-batch culture V. MnSO ₄ ions were added to induce growth of the cells in the form of pellets. That is, batch culture was performed using 20 g / L glucose and 10 g / L CSP, and then fed-batch culture was performed from 48 hours. The pH during the entire incubation period was maintained at 5.5-6.0 by adding 14% ammonia solution. Subsequently, the substrate of carbon source was supplied with 32 L of glucose solution of 750 g / L divided by 8 L (6 kg glucose) four times so that the final glucose concentration was 100 g / L. MnSO ₄ ions were added 50 mg / L at the beginning of the culture, and additionally 50 mg / L of MnSO ₄ was additionally added with glucose at the time of 72 hours of culture in which the growth of the cells was somewhat increased. Experimental results (FIG. 6) The cell mass was 44.5 g / L and the total lipid content in the cells was 60.0%, resulting in 26.7 g / L total lipid. The content of arachidonic acid in total lipids was 32.5%, yielding 8.7 g / L arachidonic acid. The supply of 14% ammonia was maintained at a culture pH of 5.5 up to 72 hours after 72 hours of incubation, and after 72 hours of incubation, the culture pH was adjusted to 6.0 or higher for about 16 hours. L 14% ammonia solution was used. The cell morphology was mixed with pellets. The results of monitoring the glucose concentration, pH, dissolved oxygen, dry weight, total lipids, arachidonic acid production, etc. over time of the incubation process of Example 6 were shown graphically in FIG. 6.

<Example 7: Fed-batch culture Ⅶ: addition of MnSO ₄ ions (2)>

A fed-batch culture was performed using a 500L fermenter at a concentration of 150 g / L of final glucose. The initial medium was subjected to batch culture with glucose 20 g / L CSP 10 g / L and fed-batch culture from 48 hours. During the culture, the pH was maintained at 5.5 to 6.0 using a 14% ammonia solution, and the sugar supply was divided into 8 L (6 kg glucose) divided by 32 L of a glucose solution of 750 g / L from 48 hours and fed 4 times up to 120 hours. From 144 hours, 16 L glucose solution of 468 g / L was supplied twice in 8 L (7.5 kg glucose) so that the concentration of sugar was finally 150 g / L. In addition, the concentration of MnSO ₄ was added 50 mg / L at the beginning of the culture, and 50 mg / L was added additionally at 48 hours when the growth of the cells was increased, and the sugar concentration was 100 g / L. 50 mg / L was added again at 120 hours to finally add 150 mg / L of MnSO ₄ . The supply of 14% ammonia was continuously supplied throughout the culture, giving a total of 20 L of ammonia. In the experimental results (FIG. 7), the cell mass was 55.5 g / L, and the total lipid content was 65.5% to produce 36.4 g / L of total lipid. In addition, the arachidonic acid content in the total lipid was 41.5%, which produced 15.1 g / L of arachidonic acid. During the entire culture period, the cells grew in the form of pellets. The results of monitoring the glucose concentration, pH, dissolved oxygen, dry weight, total lipids, arachidonic acid production, etc. over time of the incubation process of Example 7 are shown graphically in FIG. 7.

By using the arachidonic acid production method according to the present invention, it is possible to mass-produce arachidonic acid at low cost by forming a culture condition in which the genus Mortierella strain can produce a large amount of arachidonic acid. In addition, there is an effect that can be incubated in a high concentration of a small pellet (pellet) state containing arachidonic acid and lipid.

Claims (9)

In culturing Mortierella sp. , Using glucose as a carbon source and cornstalk powder as a nitrogen source, Whenever the pH falls below a specific pH within the range of pH 5 to 6, 7-40% of ammonia solution is supplied to the fermenter to adjust the pH, The ammonia solution supplied to the fermenter is a method for producing arachidonic acid through oil culture of the genus Mortierella, characterized in that also used as a nitrogen source. According to claim 1, wherein the specific pH is a method for producing arachidonic acid through the fertilization culture of the genus Mortierella, characterized in that 5.5. According to claim 1, wherein the specific pH is 5.5 up to a specific time in the range of 68 to 76 hours after the culture, and after the specific time production of arachidonic acid through the fermentation culture of the genus Mortierella strain, characterized in that 6.0 Way. The method of claim 1, wherein the cultivation is started by using a medium culture of mortiella sp. Production method of arachidonic acid. According to claim 1, wherein the culture is in the middle of the culture, when the concentration of the initially supplied glucose is less than 15g / L, the additional glucose is supplied to the fermentor, arachidone through fed-value culture of the genus Mortierella strains Acid production method. According to claim 1, The culture is in the middle of cultivation, MnSO ₄ is added to the fermentor to induce the growth of the cells in pellet form while increasing the content of arachidonic acid in the lipids, characterized in that the fermentation of strains of the genus Mortierella Production method of arachidonic acid through. The method of claim 1, wherein the culturing changes the stirring speed to maintain dissolved oxygen in the culture medium and induces cell growth in pellet form. The method of claim 1, wherein the Mortierella genus strain is a strain belonging to Mortierella alpina . The method of claim 8, wherein the genus Mortierella strain is Mortierella alpina DSA-12.