KR101068520B1 - Microorganism producing omega-3 unsaturated fatty acid having high amount of docosahexaenoic acid and processes for preparing omega-3 unsaturated fatty acid using the same - Google Patents

Abstract

The present invention comprises the step of treating curcumin (curcumin) in the microalgae (microalgae) having the ability to produce omega-3 unsaturated fatty acids, separating the strain having the ability to produce omega-3 unsaturated fatty acids and curcumin, omega- A method for producing a strain having a high content of docosahexaenoic acid in triunsaturated fatty acids; And Schizochytrium sp. Having omega-3 unsaturated fatty acid producing ability and curcumin resistance obtained from the preparation method . Schizochytrium sp. CC44 (KCTC 11566BP) is provided. In addition, the present invention comprises the steps of culturing the strain obtained by the method for producing the strain; And it provides a method for producing an omega-3 unsaturated fatty acid comprising the step of extracting the omega-3 unsaturated fatty acid from the obtained cells (biomass) or culture.

Curcumin, Schizochytrium, Docosahexaenoic Acid, Omega-3 Unsaturated Fatty Acids

Description

Microorganism producing omega-3 unsaturated fatty acid having high amount of docosahexaenoic acid and processes for preparing Strains for producing omega-3 unsaturated fatty acids containing high content of docosahexaenoic acid omega-3 unsaturated fatty acid using the same}

The present invention relates to a strain for producing an omega-3 unsaturated fatty acid containing a high content of docosahexaenoic acid and a method for producing an omega-3 unsaturated fatty acid using the same.

Omega-3 unsaturated fatty acids are also called omega-3 highly unsaturated fatty acids, typically docosahexaenoic acid (DHA) and eicosapenta. Esansapentaenoic aicd (EPA). Arachidonic acid (ARA), Docosapentaenoic acid (DPA), α-linolenic acid, and the like are known. Docosahexaenoic acid and eicosapentaenoic acid are essential fatty acids that are not synthesized in the body and should be consumed mainly through food.

Docosahexaenoic acid is abundant in the retina, semen and brain tissue of humans and animals, and is an essential fatty acid that makes up 60% of brain fat. Docosahexaenoic acid, along with arachidonic acid (ARA), is known to be important for the healthy development of the brain, eyes and nervous system of infants. In recent years, it has been reported to be effective in the prevention and treatment of a number of diseases ranging from cancer to arthritis, cardiovascular diseases and mental disorders (Pak Deok-cheon, Food World, 5 , 87-93 (2004)).

The main source of omega-3 unsaturated fatty acids, including docosahexaenoic acid, is fish oil derived from fish such as herring, salmon and tuna. However, there is a problem of continuous supply of fish oil and contamination by heavy metals and organic chemicals of fish oil, and research on the preparation method of omega-3 unsaturated fatty acid including docosahexaenoic acid by microbial culture is in progress.

Regarding the production of omega-3 unsaturated fatty acids using microorganisms, the production method of omega-3 unsaturated fatty acids by the microorganisms of the genus Thraustochytrium and Schizochytrium , which is a kind of marine microalgae, is known as Ellenbogen et al. BB, S. Aaronson, S. Goldstein and M. Belsky, Comparative Biochemistry and Physiology, 29 , 805-811 (1969).

In addition, Martek (Shizoquitririum sp. Schizochytrium sp. ATCC 20888 and Schizochytrium sp. A method for preparing omega-3 unsaturated fatty acids using ATCC 20889 ( Schizochytrium sp. ATCC 20889) has been disclosed (US Pat. No. 5,130,242 and US Pat. No. 5,340,742). In addition, Suntory has reported Schizochytrium limacinum SR21 as a microorganism having excellent docosahexaenoic acid productivity (Japanese Patent Laid-Open No. 1997-000284, US Pat. No. 6,582,941). .

In addition, in the conventional method for producing omega-3 unsaturated fatty acid using microorganisms, in order to grow microorganisms, the medium should contain seawater or a large amount of sodium chloride, which are difficult to control the components. The inventors of the present invention have problems in the conventional manufacturing method, that is, production control strains capable of effectively producing omega-3 unsaturated fatty acids without the use of seawater or sodium chloride, which are difficult to control the components and cause corrosion of the fermenter. Schizochytrium sp. RT0100P1 ( Schizochytrium sp. RT0100P1) (KCTC 10937BP)] was isolated and developed a method for producing omega-3 unsaturated fatty acid using this (Korea Patent Registration No. 10-0700486)

In addition, as a method of strain improvement for the production of omega-3 unsaturated fatty acid, a method for producing eicosapentaenoic acid by expressing the eicosapentaenoic acid synthase in E. coli (US Pat. No. 5,683,898); A method of producing polyunsaturated fatty acids by expressing polyketide synthase in plants (US Pat. No. 6,140,486); A method of increasing the production of polyunsaturated fatty acids by expressing elongase, desaturase with increased activity in fatty acid producing yeast (US Pat. No. 7,125,672); Method of producing polyunsaturated fatty acids by expressing delta 6, delta 9, delta 12, NADH-cytochrome b5 in the fungus Aspergillus (Sakuradani E, Kobayashi M, Shimizu S, 1999, Eur J Biochem, Gene, A strain improvement method by genetic engineering such as Appl Environ Microbiol) has been reported. However, the development of strains by genetic manipulation and mutation treatment of microalgae producing docosahexaenoic acid has not been reported so far. This is because microalgae biosynthesis pathways are not fully understood, so it is difficult to find a target for genetic manipulation or mutant treatment, and because microalgae are higher organisms than general bacteria, restoration systems for mutants such as NTG have been developed. It is presumed that the development of strain through mutation is difficult.

The present inventors have carried out various studies to develop strains that can further increase the content of docosahexaenoic acid in omega-3 unsaturated fatty acids. In particular, the present inventors have tried to improve the strain by treating curcumin in the microalgae having the ability to produce omega-3 unsaturated fatty acids, paying attention to the characteristics of curcumin acting as an antagonist in fatty acid biosynthesis metabolism. Surprisingly, the inventors have discovered that strains with curcumin resistance can be isolated by treating curcumin, and the isolated strains produce omega-3 unsaturated fatty acids with high content of docosahexaenoic acid.

Accordingly, an object of the present invention is to provide a method for producing a strain that produces an omega-3 unsaturated fatty acid having a high content of docosahexaenoic acid from a microalgae having an omega-3 unsaturated fatty acid producing ability.

In addition, an object of the present invention is to provide a strain produced by the method for producing the strain, to produce an omega-3 unsaturated fatty acid having a high content of docosahexaenoic acid.

In addition, an object of the present invention is to provide a method for producing an omega-3 unsaturated fatty acid having a high content of docosahexaenoic acid by culturing a strain obtained according to the method for producing the strain.

According to one aspect of the invention, (a) treating curcumin in a microalgae having an omega-3 unsaturated fatty acid production capacity, (b) omega-3 unsaturated fatty acid production capacity and curcumin resistance There is provided a method for producing a strain having a high content of docosahexaenoic acid in the omega-3 unsaturated fatty acid, comprising the step of separating the strain having.

In the preparation method of the strain according to the present invention, the microalgae is Schizochytrium strain, Thraustochytrium strain, Japonochytrium strain, Ulkenia genera Strain, Crypthecodinium may be a strain selected from the group consisting of, preferably Schizochytrium sp. RT0100P1 ( Schizochytrium sp. RT0100P1) (KCTC 10937BP) or Schizochytrium sp. SEK-228 ( Schizochytrium sp. RT0100P1) (KCTC 10938BP), more preferably Schizochytrium sp. Schizochytrium sp. RT0100P1 (KCTC 10937BP). In the above production method, step (a) adds N-methyl-N-nitro-N-nitroguanidine to microalgae having the ability to produce omega-3 unsaturated fatty acids to induce mutations, and then curcumin to treat a single colony. It can be preferably performed by selecting.

According to another aspect of the present invention, Schizochytrium sp. Having omega-3 unsaturated fatty acid producing ability and curcumin resistance . Schizochytrium sp. CC44 (KCTC 11566BP) is provided.

According to another aspect of the invention, (a ') culturing the strain obtained by the method for producing the strain; And (b ') extracting an omega-3 unsaturated fatty acid from a biomass or a culture solution obtained in step (a').

In the method for producing omega-3 unsaturated fatty acid according to the present invention, the strain is Schizochytrium sp. Schizochytrium sp. CC44 (KCTC 11566BP). The step (a ') may be carried out in a medium containing a carbon source and a nitrogen source and free of seawater and sodium chloride, wherein the carbon source is selected from the group consisting of glucose, fructose, galactose, glycerol, and mixtures thereof, and The nitrogen source may be selected from the group consisting of sodium glutamate, peptone, tryptone, yeast extract, corn steep liquor, sodium nitrate, ammonium sulfate, or mixtures thereof. In addition, the carbon source may be 2 to 20% (w / v) with respect to the total medium, and the nitrogen source may be 0.1 to 5% (w / v) with respect to the total medium.

In addition, the extraction solvent of step (b ') may be selected from the group consisting of pentane, hexane, heptane, cyclo hexane, toluene, methylene chloride, methyl acetate, acetone, chloroform, methanol, and a mixed solvent thereof.

According to the present invention, it has been found that strains can be improved by treating curcumin with microalgae, in particular omega-3 unsaturated fatty acid-producing ability, in which a restoration system for a mutant such as NTG is developed. When curcumin resistant strains, i.e., strains having omega-3 unsaturated fatty acid producing ability and curcumin resistance, were cultured, it was found that the content of docosahexaenoic acid in the omega-3 unsaturated fatty acids obtained was greatly increased. In addition, the Schizochytrium sp. Obtained by the curcumin treatment . CC44 ( Schizochytrium sp. CC44) (KCTC 11566BP) has the ability to produce omega-3 unsaturated fatty acids with a high content of docosahexaenoic acid. That is, the strain has a high proportion of docosahexaenoic acid in the total fatty acids and high docosahexaenoic acid content per cell to enable industrial production of high value added docosahexaenoic acid.

Curcumin is known to inhibit the activity of Acetyl-CoA carboxylase (US Patent Publication No. 2005/0267221). Acetyl-CoA carboxylase is an enzyme that converts acetyl-CoA to malonyl-CoA, and acetylco-CoA and malonyl-CoA are basic precursors for fatty acid biosynthesis. In particular, malonyl-CoA is used as a basic structural unit used to increase the carbon chain of fatty acids. The fatty acid biosynthesized from the basic precursor is used as a material or storage energy to form a cell membrane. Curcumin is also known to inhibit the activity of δ-5 desaturase and δ-6 desaturase, which are involved in the biosynthesis of unsaturated fatty acids (Shimizu S, 1992, Lipids). δ-6 desaturase is an enzyme that converts linolic acid to γ-linolenic acid and δ-5 desaturase It is an enzyme that converts eicosatetraenoic acid to eicosapentaenoic acid (EPA).

The present inventors paid attention to the characteristics of the curcumin, and tried to improve the strain by treating the microalgae (microalgae) having the ability to produce omega-3 unsaturated fatty acids with curcumin. Surprisingly, the present inventors have found that microalgae in which a recovery system for NTG and the like has been developed can be improved by treating curcumin, and the resulting curcumin-resistant strain, i.e., omega-3 unsaturated fatty acid producing ability When culturing strains with curcumin resistance, it was found that the content of docosahexaenoic acid in the omega-3 unsaturated fatty acids obtained increased significantly.

Therefore, the present invention comprises the steps of: (a) treating curcumin in a microalgae having an omega-3 unsaturated fatty acid producing ability, (b) isolating a strain having an omega-3 unsaturated fatty acid producing ability and curcumin resistance It provides a method for producing a strain having a high content of docosahexaenoic acid in the omega-3 unsaturated fatty acid, comprising the step of.

In the preparation method, the microalgae are Schizochytrium strains, Thraustochytrium strains, Japonochytrium strains, Ulkenia strains, and Crypcocordi . It may be a strain selected from the group consisting of strains of the genus Crypthecodinium , preferably Schizochytrium sp. RT0100P1 ( Schizochytrium sp. RT0100P1) (KCTC 10937BP) or Schizochytrium sp. SEK-228 ( Schizochytrium sp. RT0100P1) (KCTC 10938BP), more preferably Schizochytrium sp. Schizochytrium sp. RT0100P1 (KCTC 10937BP).

Step (a) is preferably performed by adding N-methyl-N-nitro-N-nitroguanidine to the microalgae having the ability to produce omega-3 unsaturated fatty acids to cause mutation, and then treating curcumin to select a single colony. Can be.

Specifically, step (a) is a microalgae having an omega-3 unsaturated fatty acid producing ability, for example, Schizochytrium sp. After culturing RT0100P1 ( Schizochytrium sp. RT0100P1) (KCTC 10937BP) in a medium such as GPY medium and adding NTG (N-methyl-N-nitro-N-nitroguanidine) to induce mutation, curcumin is It can be performed by screening a single colony by spreading to a selective medium containing 50 to 200 mg / L concentration (eg, OF-2 plate sorting medium). The GPY medium may be composed of 20 g / L glucose, 10 g / L peptone, 5 g / L yeast extract, and 50% artificial seawater (20 g / L sea salt from Sigma), but is not limited thereto. In addition, the OF-2 medium is glucose 20 g / L, sodium glutamate 10 g / L, ammonium sulfate 2 g / L, potassium chloride 1 g / L, magnesium sulfate (MgSO ₄ 7H ₂ O) 10 g / L, hydrogen carbonate Sodium 0.2 g / L, potassium dihydrogen phosphate 5 g / L, trace component I 5 ml / L, trace component II 5 ml / L, calcium chloride 3 g / L, and agar 20 g / L, but It is not limited. In addition, if necessary, the culture solution obtained by culturing in GPY medium before the NTG treatment may be centrifuged and washed, and the cell concentration may be adjusted to a predetermined level, for example, 10 ⁶ to 10 ⁸ / ml, followed by NTG treatment. have.

Step (b) may be carried out by culturing the strain selected from step (a) in a production medium such as OF-12 medium, and separating the strain having a high content of docosahexaenoic acid in the total fatty acids. The OF-12 medium is glucose 120 g / L, sodium glutamate 10 g / L, ammonium sulfate 2 g / L, potassium chloride 1 g / L, magnesium sulfate (MgSO ₄ 7H ₂ O) 10 g / L, sodium hydrogencarbonate 0.2 g / L, potassium dihydrogen phosphate 5 g / L, trace component I 5 ml / L, trace component II 5 ml / L, and calcium chloride 3 g / L, but are not limited thereto.

In the medium component, the trace component I is 200 mg / L thiamine hydrochloride, 0.5 mg / L biotin, vitamin B ₁₂ 50 μg / L, nicotinic acid 100 mg / L, calcium pantothenate 100 mg / L, riboflavin 5 mg / L, hydrochloric acid Pyridoxine 40 mg / L, paraaminobenzoic acid 10 mg / L, inositol 1000 mg / L, orotic acid 260 mg / L, folic acid 2.5 mg / L (pH 7.2); Trace ingredient Ⅱ is 6 g / L sodium idieti, 0.29 g / L iron chloride (FeCl ₃ 6H ₂ O), 6.84 g / L boric acid, 0.86 g / L manganese chloride (MnCl ₂ 4H ₂ O), zinc chloride (ZnCl ₂ ) 0.06 g / L, cobalt chloride (CoCl ₂ 6H ₂ O) 0.026 g / L, copper sulfate (CuSO ₄ 5H ₂ O) 0.002 g / L (pH 7.2).

The present invention also provides an improved strain obtained according to the method for producing the strain. In particular, the present inventors have isolated a variety of mutant strains according to the production method of the strain, of which the present inventors have developed the Schizochytrium sp. Korean Patent Registration No. 10-700486 . Variants isolated from RT0100P1 ( Schizochytrium sp. RT0100P1) (KCTC 10937BP) were isolated from those with significantly higher docosahexaenoic acid content in omega-3 unsaturated fatty acids. Among the mutant strains, the strain having the highest content of docosahexaenoic acid among the total fatty acids was Schizochytrium sp. It was named CC44 ( Schizochytrium sp. CC44), and it was deposited on September 25, 2009 to the Korea Institute of Bioscience and Biotechnology, Genetic Bank (KCTC), and received an accession number of KCTC 11566BP. Thus, in one embodiment, the present invention provides an omega-3 unsaturated fatty acid producing ability and curcumin resistance to Schizochytrium sp. Schizochytrium sp. CC44 (KCTC 11566BP) is provided.

The invention also (a ') culturing the strain obtained by the method for producing the strain; And (b ') extracting an omega-3 unsaturated fatty acid from a biomass or a culture solution obtained in step (a').

In the method for producing omega-3 unsaturated fatty acid according to the present invention, the strain is Schizochytrium sp. Schizochytrium sp. CC44 (KCTC 11566BP).

In the production method of the present invention, the step of culturing the strain is preferably carried out in a medium containing a carbon source and a nitrogen source, and does not contain sea water and sodium chloride, the carbon source is glucose, fructose, galactose, glucose, glycerol, And mixtures thereof, wherein the nitrogen source is selected from the group consisting of sodium glutamate, peptone, tryptone, yeast extract, corn steep liquor, sodium nitrate, ammonium sulfate, ammonium citrate, or mixtures thereof Can be. The carbon source may be contained at a concentration of 2-20% (w / v), preferably 9-15% (w / v), in the total medium, and the nitrogen source may be 0.1-5% (w / v) in the total medium. ), Preferably 1 to 2.5% (w / v).

The production method of the present invention includes the step of extracting the omega-3 unsaturated fatty acid from the obtained biomass or culture medium. The extractant may be selected from the group consisting of pentane, hexane, heptane, cyclohexane, toluene, methylene chloride, methyl acetate, acetone, chloroform, methanol, and a mixed solvent thereof, preferably hexane. .

In the extraction step, the supernatant is removed by centrifugation of the culture solution to recover the cells, and the recovered cells are preferably reduced to about 5.0% or less by a drying method such as spray drying, and, if necessary, to prevent rancidity. You can also add an agent. The amount of the extraction solvent is preferably used in a ratio of about 5 L to 1 kg of dry cells.

The extraction process is a conventional method, that is, the homogenizing and adding the extraction solvent, the cell can be crushed to extract the omega-3 unsaturated fatty acid. After extraction, the cell lysate is removed by centrifugation, and the extraction solvent is removed by distillation under reduced pressure, thereby obtaining an omega-3 unsaturated fatty acid in a crude state. At this time, the extraction solvent can be recovered and reused.

The production method of the present invention may include a conventional purification process. That is, by carrying out the winterization process in accordance with a known method (US Pat. No. 6,812,009) can be carried out to remove the saturated fatty acid, and also, according to the known method (US Pat. No. 6,750,048) degum, It is possible to remove phospholipids, free fatty acids, pigments, remaining saponified substances, low molecular volatiles causing odors by carrying out a fat or oil manufacturing process such as neutralization, decolorization and deodorization.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are intended to illustrate the invention, but not to limit the scope of the invention.

Example 1. Selection and Cultivation of Mutant

Schizochytrium sp. After culturing RT0100P1 ( Schizochytrium sp. RT0100P1) (KCTC 10937BP) in GPY medium for 24 hours at 13000xg, 0.05M TM buffer solution (0.2M Trizma base, 0.2M maleic acid) (Maleic acid), 0.2M NaOH, pH6.0) were added twice in the same volume and washed twice. After diluting the cell concentration to 10 ⁶ ~ 10 ⁸ / ml with the buffer, 200 μg / ml NTG solution was treated for 20 minutes at 30 ℃. The treated cells were washed twice with physiological saline, and then plated on OF-2 plate medium to which curcumin 100 mg / L was added and allowed to stand at 25 ° C for 5 days. Strains forming a single colony were again passaged twice on OF-2 plate medium supplemented with curcumin 100 mg / L. The treatment was carried out as described above to obtain a total of 254 curcumin resistant strains, each of the mutants were cultured to produce omega-3 unsaturated fatty acids as follows.

10 ml of GPY medium was dispensed into 100 ml Elenmeyer flasks to inoculate each strain and shaken for 24 hours at 140 rpm at 25 ° C. as a seed culture. 30 ml of OF-12 medium was dispensed into a 300 ml Elenmeyer flask, inoculated with 0.6 ml of the previously prepared seed culture, and incubated for 96 hours at 25 rpm at 140 rpm. The strains showing the highest content of docosahexaenoic acid were isolated by measuring the proportion of docosahexaenoic acid in the total fatty acids of each flask culture . It was named CC44 ( Schizochytrium sp. CC44).

The Schizochytrium sp. Flask culture results for RT0100P1 and CC44 and the results of measuring the ratio of each fatty acid in the total fatty acids are shown in Table 1 and Table 2. After incubation, the glucose concentration was analyzed by YSI Model 2700 SELECT (YSI, USA), and the dry cell weight was measured by washing the culture solution and drying it at 105 ° C. In addition, the cells cultured as described above were collected by centrifugation, extracted with Folch solution (chloroform: methanol = 2: 1, volume ratio) for 1 hour at 30 ° C. and methylated with Meth-Prep II of Alltech, followed by HP gas. The concentration of docosahexaenoic acid was calculated using chromatography (Medel 6890), and the component ratio of each fatty acid was calculated using the area ratio of the peak having the same retention time as the standard fatty acid, and the results are shown in Tables 1 and 2 below. .

Flask culture results Strain Incubation time
(hrs) Remnant
(%) DHA
(g / L) DHA in fatty acids (%) Cell weight
(g / L) DHA per cell (%) DHA / ΔRS DCW / ΔRS RT0100P1 96 0.2 6.3 20 52.2 12.1 0.53 4.43 CC44 146 0.3 10.6 50 44.3 24.0 0.91 3.79

※ DHA (%) among fatty acids: The value calculated based on the area during GC analysis, and the ratio of DHA peak area to the total fatty acid peak area.

The ratio of each fatty acid out of total fatty acids Strain DHA DPA C14
Myristic acid
(Myristic acid) C15 C16
Palmitic acid
(Palmitic acid) C17 PUFA
(DHA + DPA) RT100P1 20 7 26 3 38 0 27 CC44 50 17 One 8 15 5 67

※ Fatty acid ratio is calculated based on area during GC analysis

From the results of Table 1 and Table 2, Schizochytrium sp. CC44 ( Schizochytrium sp. CC44) has a Docosahexaenoic acid ratio of 50% or more of the total fatty acids, the concentration of docosahexaenoic acid per culture medium is Schizochytrium sp. More than 1.6 times higher than RT0100P1 ( Schizochytrium sp. RT0100P1) (based on consumption glucose 12%), Schizochytrium sp. Compared to RT0100P1 ( Schizochytrium sp. RT0100P1), the ratio of saturated fatty acids, myristic acid and palmitic acid, is reduced, resulting in the addition of docosahexaenoic acid and docosapentaenoic acid in total fatty acids. It can be seen that the proportion of unsaturated fatty acids is 65% or more.

Example 2. Comparison of Resistance to Curcumin

50 ml of GPY medium was dispensed into 500 ml Elenmeyer flasks to inoculate strains, and shaken at 25 ° C. for 24 hours at 140 rpm to obtain a seed culture. Curcumin was added to 50 ml of GPY medium so as to be 0, 50 and 75 100 mg / L, respectively, and the cells were dispensed into 500 ml Elenmeyer flasks, inoculated with 1 ml of the previously prepared seed culture solution, and incubated at 28 ° C. at 200 rpm for 24 hours. The growth of the culture medium without curcumin was 100 and the growth of the culture medium with curcumin was shown as relative growth . CC44 ( Schizochytrium sp. CC44) is Schizochytrium sp. Compared to RT0100P1 ( Schizochytrium sp. RT0100P1), resistance to curcumin was significantly increased (see Table 3).

Curcumin
(mg / L) Relative Growth (%) RT0100P1 CC44 0 100.0 100.0 50 18.3 45.0 75 8.6 25.6 100 5.0 15.6

Example 3 Comparison of Docosahexaenoic Acid Ratio Changes in Culture

Schizochytrium sp. CC44 ( Schizochytrium sp. CC44) and Schizochytrium sp. RT0100P1 ( Schizochytrium sp. RT0100P1) flasks production medium OF-9 (glucose 90 g / L, sodium glutamate 10 g / L, ammonium sulfate 2 g / L, potassium chloride 1 g / L, magnesium sulfate (MgSO ₄ · 7H ₂ O) 10 g / L, 0.2 g / L sodium hydrogencarbonate, 5 g / L potassium dihydrogen phosphate, 5 ml / L of trace component I, 5 ml / L of trace component II, and 3 g / L of calcium chloride) Docosahexaenoic acid ratio change was analyzed according to the results, as shown in Table 4. Schizochytrium sp. RT0100P1 ( Schizochytrium sp. RT0100P1) shows that the rate of docosahexaenoic acid decreases as the docosahexaenoic acid is 55% in the lag phase of the initial culture, but as the log phase grows. As a result, the docosahexaenoic acid ratio was maintained at 19% at 8.8%. But the Schizochytrium sp. CC44 ( Schizochytrium sp. CC44) showed that the rate of docosahexaenoic acid among total fatty acids was 55% in the early stage of incubation, similar to RT0100P1, but the rate of docosahexaenoic acid was maintained even when the growth was progressed through logarithmic growth. Even at 7.9% of sugar, the rate of docosahexaenoic acid was 53%, indicating that fatty acid biosynthesis was different from that of parent. The ratio of docosahexaenoic acid in the total fatty acids is a unique characteristic of the strain, which is also a criterion for classification of the species.

Changes in Docosahexaenoic Acid Ratio in Culture Strain Incubation time
(hrs) Consumed sugar
(%) Cell weight
(g / L) DHA
(g / L) DHA among fatty acids
(%) DHA per cell
(%) RT0100P1 24 0.4 7.86 0.21 55 2.7 48 4.4 22.05 1.81 24 8.2 66 8.8 29.52 3.5 19 11.9 CC44 24 One 10.72 0.94 55 8.8 48 5.5 22.82 4.29 52 18.8 64 7.9 27.3 5 53 18.3

Example 4. Strain Comparison Using Electron Microscopy

Schizochytrium sp. RT0100P1 ( Schizochytrium sp. RT0100P1) and Schizochytrium sp. Schizochytrium sp. CC44 and the previously reported docosahexaenoic acid producing strain Schizochytrium limacinum SR21, Schizochytrium sp. It was measured using SEM and TEM to compare the size and cell wall thickness of the strain of ATCC 20888 ( Schizochytrium sp. ATCC 20888). The results are shown in FIGS. 1 to 3. Schizochytrium sp. RT0100P1 ( Schizochytrium sp. RT0100P1) and Schizochytrium sp. CC44 ( Schizochytrium sp. CC44) was found to be larger in size than Schizochytrium limacinum SR21 ( Sizochytrium limacinum SR21) having a cell size of 10 to 20 ㎛ 5-10 ㎛ (see Figure 1). In addition, Schizochytrium limacinum SR21 ( Schizochytrium limacinum SR21) is present alone without forming a pellet, but Schizochytrium sp. RT0100P1 ( Schizochytrium sp. RT0100P1) and Schizochytrium sp. CC44 ( Schizochytrium sp. CC44) was present in pellet form (see FIG. 2). Schizochytrium sp. The cell wall thickness of RT0100P1 ( Schizochytrium sp. RT0100P1) was very diverse, ranging from 44 to 637 nm . CC44 ( Schizochytrium sp. CC44) was 50-121 nm. The cell wall thickness of Schizochytrium limacinum SR21 is 7-83 nm, and Schizochytrium sp. The cell wall thickness of ATCC 20888 ( Schizochytrium sp. ATCC 20888) was 22-315 nm (see FIG. 3).

Example 5 Nitrogen Source Availability Comparison

In order to analyze the growth of each DHA-producing strain according to the nitrogen source, 10 ml of GPY medium was dispensed into a 100 ml Elenmeyer flask to inoculate the strain, and cultured at 25 ° C. for 24 hours at 140 rpm to obtain a seed culture. Nitrogen source availability test medium is shown in Table 5. Ten milliliters of a nitrogen-source-available medium, each containing 12 different nitrogen sources at a concentration of 2 g / L, were dispensed into a 100 ml Elenmeyer flask and inoculated with 0.2 ml of the previously prepared seed culture medium at 5 ° C. at 140 rpm for 5 days. After shaking culture, the growth of bacteria was confirmed.

Nitrogen Source Usability Test Medium Medium Concentration (g / L) Glucose 20 NaCl 25 KCl One MgSO ₄ 7H ₂ O 5 NaHCO ₃ 0.1 KH ₂ PO ₄ 0.1 Trace Component I 5 ml CaCO ₃ 2

Schizochytrium sp. CC44 ( Schizochytrium sp. CC44) is Schizochytrium sp. Compared to RT0100P1 ( Schizochytrium sp. RT0100P1), there was a difference in the availability of nitrogen sources such as peptone and ammonium citrate (see Table 6).

Nitrogen Source Availability Comparison Growth RT0100P1 CC44 Sodium Glutamate ++ ++ peptone +++ + Trypton ++ + - Yeast extract ++ ++ Malt Extract - - Ammonium Citrate + - Ammonium Sulfate + + Ammonium acetate - - Ammonium Nitrate - - Ammonium chloride - - Sodium nitrate ++ + Urea - - Control group (without nitrogen source) - -

+: Excellent growth-: Minor growth

Example 6. 50 L Fermenter Culture

Schizochytrium sp. OF-4 medium (40 g / L glucose, 10 g / L sodium glutamate, 2 g / L ammonium sulfate, 1 g / L potassium chloride, magnesium sulfate (MgSO) in 50 L fermenter for mass cultivation of CC44 ( Schizochytrium sp. CC44) _{4 · 7H 2 O) 10 g} / L, 0.2 g / L sodium hydrogen carbonate, potassium dihydrogen phosphate 5 g / L, trace elements ⅰ 5 ml / L, trace elements ⅱ 5 ml / L, calcium chloride 3 g / L) 21 L was used and incubated at 200 rpm, 28 ° C, and 1.0 vvm. With glucose remaining in the medium at a concentration of 10 g / L, 1500 ml of a glucose solution having a glucose concentration of 380 g / L was added five times to give a total glucose concentration of 129 g / L. Cultured cells were collected in the same manner as in Example 1 to analyze the dry cell mass and fatty acids, the results are shown in Table 7.

50 L fermenter culture Strain Schizochytrium sp. CC44 Incubation time (hrs) 160 Dry cell weight (g / L) 58.5 DHA concentration per culture (g / L) 12.4 DHA concentration per dry cell (%) 21.2 % DHA in fatty acids 51

Example 6 Extraction and Purification of Omega-3 Unsaturated Fatty Acids

Fermentation was carried out in the same manner as in Example 5, and the obtained fermentation broth was centrifuged and spray dried. Hexane is added to the obtained dry cells, homogenized, and the cells are crushed to extract omega-3 unsaturated fatty acids. The amount of hexane used was used at a rate of 5 L with respect to 1 kg of dry cells, and after extraction, the cells were removed by centrifugation, and hexane was removed by distillation under reduced pressure. In accordance with US Pat. No. 6,812,009 and US Pat. No. 6,750,048, dewaxing and fat preparation processes were performed to purify omega-3 unsaturated fatty acids.

1 is a result of TEM measurement of docosahexaenoic acid producing strains, (A) Schizochytrium limacinum SR21, (B) is a Schizochytrium sp. ATCC 20888 ( Schizochytrium sp. ATCC 20888), (C) is Schizochytrium sp. RT0100P1 ( Schizochytrium sp. RT0100P1), and (D) are Schizochytrium sp. The measurement results of the CC44 (Schizochytrium sp. CC44), respectively.

Figure 2 is a SEM measurement of docosahexaenoic acid producing strains, (A) Schizochytrium limacinum SR21 (B), (B) is a Schizochytrium sp. ATCC 20888 ( Schizochytrium sp. ATCC 20888), (C) is Schizochytrium sp. RT0100P1 ( Schizochytrium sp. RT0100P1), and (D) are Schizochytrium sp. The measurement results of the CC44 (Schizochytrium sp. CC44), respectively.

Figure 3 is a TEM measurement result measured by adjusting the magnification to measure the cell wall thickness in the results of Figure 1, (A) Schizochytrium limacinum SR21 (B), (B) is a Schizochytrium sp . ATCC 20888 ( Schizochytrium sp. ATCC 20888), (C) is Schizochytrium sp. RT0100P1 ( Schizochytrium sp. RT0100P1), and (D) are Schizochytrium sp. The measurement results of the CC44 (Schizochytrium sp. CC44), respectively.

Claims (12)

(a) treating curcumin in a microalgae having an omega-3 unsaturated fatty acid producing ability, and (b) separating a strain having an omega-3 unsaturated fatty acid producing ability and curcumin resistance; , A method for producing a strain having a high content of docosahexaenoic acid in omega-3 unsaturated fatty acids. According to claim 1, wherein the microalgae are Schizochytrium strain, Thraustochytrium strain, Japonochytrium strain, Ulkenia strain, Crypsecody Method for producing a strain selected from the group consisting of strains of the genus Crypthecodinium . The method of claim 2, wherein the microalgae are Schizochytrium sp. RT0100P1 ( Schizochytrium sp. RT0100P1) (KCTC 10937BP) or Schizochytrium sp. SEK-228 ( Schizochytrium sp. RT0100P1) (KCTC 10938BP). The method of claim 3, wherein the microalgae are Schizochytrium sp. RT0100P1 ( Schizochytrium sp. RT0100P1) (KCTC 10937BP) characterized in that the manufacturing method. 2. The method of claim 1, wherein step (a) adds N-methyl-N-nitro-N-nitroguanidine to the microalgae having the ability to produce omega-3 unsaturated fatty acids to induce mutations, and then curcumin to treat a single colony. Process for producing, characterized in that carried out by screening. Schizochytrium sp. Having omega-3 unsaturated fatty acid producing ability and curcumin resistance . Schizochytrium sp. CC44 (KCTC 11566BP). (a ') culturing the strain obtained by the method according to any one of claims 1 to 5; And (b ') extracting the omega-3 unsaturated fatty acid from the biomass or the culture medium obtained in step (a'). 8. The method of claim 7, wherein said strain is Schizochytrium sp. CC44 (Schizochytrium sp. CC44) method, characterized in that (KCTC 11566BP). 9. A process according to claim 8, wherein step (a ') is carried out in a medium comprising a carbon source and a nitrogen source and free of sea water and sodium chloride. The method of claim 9, wherein the carbon source is selected from the group consisting of glucose, fructose, galactose, glycerol, and mixtures thereof, and the nitrogen source is sodium glutamate, peptone, tryptone, yeast extract, corn steep liquor, sodium nitrate, sulfuric acid Ammonium, or a mixture thereof. The method according to claim 10, wherein the carbon source is 2-20% (w / v) with respect to the total medium, and the nitrogen source is 0.1-5% (w / v) with respect to the total medium. The method of claim 7, wherein the extraction solvent of step (b ') is selected from the group consisting of pentane, hexane, heptane, cyclo hexane, toluene, methylene chloride, methyl acetate, acetone, chloroform, methanol, and a mixed solvent thereof Manufacturing method characterized in that.

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