KR101541521B1 - Method of producing microalgal dha using microalgae - Google Patents

Abstract

The present invention relates to a method for producing docosahexaenoic acid (DHA) from microalgae, and more particularly, to a method for producing docosahexaenoic acid (DHA) from microalgae comprising the steps of (a) culturing microalgae in a medium containing a carbon source and a nitrogen source, and And feeding the medium with a composition comprising a carbon source without a nitrogen source. The present invention also relates to a method for producing docosahexaenoic acid from microalgae.

Description

TECHNICAL FIELD The present invention relates to a method for producing docosahexaenoic acid using microalgae,

The present invention relates to a method for producing docosahexaenoic acid (DHA) from microalgae, and more particularly, to a method for producing docosahexaenoic acid (DHA) from microalgae comprising the steps of (a) culturing microalgae in a medium containing a carbon source and a nitrogen source, and And feeding the medium with a composition comprising a carbon source without a nitrogen source. The present invention also relates to a method for producing docosahexaenoic acid from microalgae.

Omega-3 unsaturated fatty acids are also referred to as omega-3 highly unsaturated fatty acids, and typically include docosahexaenoic acid, Eicosapentaenoic acid (Eicosapentaenoic acid, EPA), arachidonic acid (ARA), docosapentaenoic acid (DPA), and? -Linolenic acid. Docosahexaenoic acid and eicosapentaenoic acid are essential fatty acids that are not synthesized in the body and should be ingested mainly through food.

Docosahexaenoic acid is abundant in retinas, seminal and brain tissues of humans and animals, and it is an essential fatty acid that constitutes 60% of brain fat in particular. Together with arachidonic acid, docosahexaenoic acid is known to be important for the healthy development of the brain, eyes and nervous system of infants. Recently, it has been reported that it is effective in the prevention and treatment of many diseases ranging from cancer to arthritis, cardiovascular diseases and mental disorders (Park, Deokcheon, Food World, 5, 87-93 (2004)).

The main source of omega-3 unsaturated fatty acids, including docosahexaenoic acid, is fish oil extracted from fish such as herring, salmon, and tuna. However, the difficulty of supplying fish oil continuously and pollution caused by heavy metals and organic chemicals in fish oil have been studied, and studies on the production method of omega-3 unsaturated fatty acids including docosahexaenoic acid by microbial culture are under way.

The production of omega-3 unsaturated fatty acids by microorganisms such as Thraustochytrium and Schizochytrium, a kind of marine microalgae, has been studied by Ellenbogen et al. (Ellenbogen BB, S. Aaronson, S. Goldstein and M. Belsky, Comparative Biochemistry and Physiology, 29, 805-811 (1969)).

A method for producing DHA using microalgae A strain producing omega-3 unsaturated fatty acid containing a high content of docosahexaenoic acid and a method for producing omega-3 unsaturated fatty acid using the same United States Patent Application Publication No. 2011-0037588 However, it may be inconvenient to continuously supply the concentrated sugar during the fed-batch culture, and there is a problem in operation cost due to external pollution and facility operation for supplying the medium in the process. In addition, although an improved production method of lipids containing polyunsaturated fatty acids by high-density culture of eukaryotic microorganisms in a fermenter (Korean National Publication No. 2003-0013368) is known, a nonalcoholic carbon source including a nitrogen source, There is a problem of the same external contamination and increased operating cost as described above.

Under these circumstances, the present inventors have found that when a composition containing a carbon source is added temporarily without containing a nitrogen source during the fermentation of a microalgae in a medium for culturing microalgae, a high content of DHA oil The present invention has been completed.

It is an object of the present invention to provide a method of producing docosahexaenoic acid from microalgae, which is capable of reducing the risk of external pollution due to continuous supply of medium during cultivation of microalgae, Method.

(A) culturing the microalgae in a medium containing a carbon source and a nitrogen source; and (b) supplying a composition containing a carbon source without containing a nitrogen source to the medium. And to provide a method for producing tetrahexaenoic acid.

In accordance with one aspect of the present invention, there is provided a method for culturing microalgae, comprising culturing microalgae in a medium containing a carbon source and a nitrogen source, and (b) To a process for producing docosahexaenoic acid from microalgae.

Preferably, step (a) may be performed for 46 to 52 hours.

Also preferably, the culture in step (a) may be carried out at a temperature of from 15 ° C to about 34 ° C.

Also preferably, the culture in step (a) may be carried out at a pH of 4 to 6. [

Also preferably, step (b) may be such that a composition containing a carbon source without containing a nitrogen source in the medium is fed at 100 to 150% (v / v) of the medium volume.

Also preferably, the concentration of the carbon source in the composition containing the carbon source without the nitrogen source in the step (b) may be 50 to 60 g / L.

Also preferably, after step (b), the microalgae can be cultured for 69 to 74 hours.

Preferably, the carbon source may be at least one selected from the group consisting of molasses, malt extract, starch, glucose, dextrin, sulfurous acid waste, fiber, whey, alkaline, alcohol, fat and oil.

Also preferably, said microalgae are selected from the group consisting of Schizochytrium, Thraustochytriales, Ulkenia, Thraustochytrium, Japonochytrium, Creptecodinium Crypthecodinium, Haliphthoros, Chlamydomonas, Aurantiochytrium, and Porphyridium. The term " a "

Also preferably, the microalgae may be pre-cultured in a medium containing glucose (glucose), yeast extract and sea salt.

Also preferably, the medium may be a medium containing glucose, CS steal, monosodium glutamate and sea salt.

Also preferably, the microalgae after the step (b) may be cultured at 20 to 28 ° C.

Also preferably, the step (b) may comprise culturing the microalgae at pH 4 to 6.

Preferably, the method further comprises the step of recovering DHA from the culture in which the microalgae are cultured.

Hereinafter, the present invention will be described in more detail.

The present invention provides a method for producing DHA from microalgae by supplying a composition containing a carbon source during the culturing of microalgae capable of producing DHA.

In a specific example of the present invention, in order to examine the effect on the production of DHA when a composition containing a carbon source is supplied during the cultivation of microalgae, comparative experiments were carried out in the case of continuous culturing under the initial conditions without supplementary medium.

As a result, it was confirmed that when the composition containing the carbon source was supplied during the cultivation of the microalgae according to the present invention, the cell concentration, DHA content per cell, DHA concentration and yield were all significantly superior One).

The present invention relates to a method for producing DHA comprising the steps of (a) culturing microalgae in a medium containing a carbon source and a nitrogen source, and (b) supplying a composition containing a carbon source without containing a nitrogen source to the medium In this culture, microbial growth and DHA production can be increased.

For example, the step (a) may be carried out for 46 to 52 hours. If the incubation time is less than 46 hours, sufficient microalgae growth may not be achieved. If the incubation time is longer than 52 hours, the microalgae growth may enter the stationary phase and the DHA production may be decreased due to low cell growth at the subsequent stage. Specifically, the step (a) may be performed for 48 to 52 hours, 49 to 51 hours, or 48 hours, but is not limited thereto, and can be appropriately adjusted according to the volume of the medium, the concentration of the microalgae in the medium, and the like.

Following step (a), step (b) may be carried out by supplying a medium containing no carbon source to the medium of step (a) in an amount of 100 to 150% (v / v) have. The DHA content may be reduced when 100% (v / v) or less of the volume of the medium is supplied, and when the amount exceeds 150% (v / v) of the volume of the medium, The growth of microalgae can be inhibited. Specifically, a composition containing a carbon source may be supplied at 100 to 120% (v / v), 100% (v / v) of the volume of the medium, but is not limited thereto.

In addition, in the step (b), the concentration of the carbon source in the composition containing no carbon source may be 50 to 60 g / L. When the concentration of the carbon source is less than 50 g / L, the DHA content may be decreased. When the concentration of the carbon source is more than 60 g / L, the yield may be decreased because the increase of the DHA accumulation amount is smaller than that of the carbon source (sugar). For example, the concentration of a carbon source in a composition that does not include a nitrogen source but contains a carbon source may be 52 to 60 g / L, 55 to 60 g / L, or 58 g / L, but is not limited thereto.

As another example, step (b) can be used to incubate microalgae for 69-74 hours. If the incubation time of the microalgae after step (b) is less than 69 hours, the DHA content may be insufficient. If the microalgae is cultured for more than 74 hours, the DHA accumulation may no longer be consumed. Specifically, the step (b) may be performed for 70 to 72 hours, 71 to 73 hours, or 72 hours, but is not limited thereto and can be appropriately adjusted according to the volume of the medium, the concentration of the microalgae in the medium, and the like.

In addition, step (b) can be used to culture the microalgae at 20 to 28 占 폚. If the incubation temperature is outside the above range, the growth of microalgae can be inhibited. Specifically, the step (b) may be carried out at 22 to 27 占 폚, 23 to 26 占 폚 or 25 占 폚, but is not limited thereto.

The term "carbon source" in the present invention means a carbon compound which is used for growth and proliferation of microalgae cultured in a medium, and is absorbed by microalgae and used as a biocompatible carbon. For example, it may be molasses, malt extract, starch, glucose, dextrin, sulfurous acid waste solution, fiber, whey, alkaline, alcohol, fat, oil and the like. Falls within the scope of the invention.

The term "nitrogen source" refers to a nitrogen compound that is a source of protein or nucleic acid in an organism. For example, inorganic nitrogen compounds, nitrates and ammonium salts, organic nitrogen compounds, nitrites, hydroxylamines, amino acids, amides, amines, CSL, Urea, casein, soybean meal, wheat bran, rice bran, But the present invention is not limited thereto, and any material that can use microalgae as a nitrogen source falls within the scope of the present invention.

In the present invention, microalgae generally include all microalgae containing DHA. Specifically, there may be mentioned a strain belonging to the genus Zizania, a genus of Trastus tritiatricus, a genus of Wolkenia, a genus of Trotsky tritium, a genus Zaponochitium, a genus Kryptocordinum, a genus of halifotros, a genus Clamidomonas, ≪ / RTI > and < RTI ID = 0.0 > Porphyridium < / RTI > To maximize DHA production, the microalgae can be an orchidychititic spider, preferably Aurantiochytrium limacinum SR21.

The culture medium for culturing the microalgae in the step (a) and the step (b) may include glucose, CSL, monosodium glutamate and sea salt. In one example, the medium may include, but is not limited to, 60 to 100 g / L of glucose and 10 to 50 g / L of CSL. Specifically, components known to promote growth and production of DHA, such as those described in, for example, U.S. Patent No. 5,397,591, U.S. Patent No. 5,492,938, and U.S. Patent No. 5,711,983, may be included. More specifically, a carbon source, affinity organic or inorganic nitrogen source may also be included in the culture medium. The nitrogen source may include nitrates, ureas, ammonium salts, amino acids, and the like. A source of assimilable phosphorous may also be provided. The medium may also contain a source of microbial growth factors, which are compounds that promote heterotrophic bio-growth of single cell microorganisms, and may also include yeast or microbial extracts, soil extracts, and the like.

The term "sea salt" is sodium chloride (NaCl), MgCl ₂ (magnesium chloride), K ₂ SO ₄ (potassium sulfate), MgSO ₄ (magnesium sulfate), potassium chloride (KCl), calcium chloride (CaCl _2), sodium bicarbonate (NaHCO ₃ ) And the like, and means a salt which can be used as a medium component. For example, artificial sea salt from Sigma-Aldrich.

In order to improve the growth of microalgae and the production of DHA in the present invention, the microalgae can be pre-cultured before performing the step (a). The medium used for the pre-culture may include, but is not limited to, glucose, yeast extract and seaweed. For example, the pregelatinized medium may include glucose 30 g / L, yeast extract 10 g / L, sea salt 27 g / L. Specifically, pre-cultivation can be carried out at 20 to 28 캜, pH 6.

When the pre-culture is performed before the main culture, the microalgae that have been lyophilized can be activated, and the present culture can be performed in a uniform state, so that DHA can be produced with excellent quality.

As a specific example, in the present invention, the microalgae can be cultured under conditions of 0.5 vvm of aeration and 350 rpm of agitation.

Other optimal conditions for the microalgae culture method can also be readily determined by those skilled in the art. In short, the culturing can be carried out in a suitable fermenter, preferably a stirred tank fermenter or an air-lift fermenter, which provides an oxygen source to the microalgae. Stirring of the microalgae should maintain the dissolved oxygen concentration sufficient to support the growth of the culture and the production of DHA, while stirring does not truncate or otherwise damage the microalgae. A preferred level of dissolved oxygen is at least 10% of the saturation concentration. More preferably, the concentration of dissolved oxygen is maintained at an air saturation concentration of from about 10% to about 50%.

The microalgae can be cultured at a specific life-sustaining temperature. Generally, the microalgae grow at a temperature of about 15 ° C to about 34 ° C. For example, the incubation temperature of the microalgae can be from about 20 ° C to about 28 ° C.

Although not particularly limited, the pH can be maintained at 4 to 6 during microalgae culture.

In another embodiment of the present invention, the method may further include the step of recovering DHA.

In more detail, the microalgae can be recovered by conventional means known to those skilled in the art, such as by centrifugation, flocculation or filtration, and can be processed immediately or dried for further processing. In each case, lipids can be extracted. The term " lipid ", as used herein, includes phospholipids, free fatty acids, fatty acid esters, triacylglycerols, diacylglycerides, monoacylglycerides, lysophospholipids, soaps, phosphatides, sterols and sterol esters, carotenoids, (E.g., oxycarotenoids), hydrocarbons, and other lipids known to those skilled in the art.

As will be appreciated by those skilled in the art, the DHA referred to in the present invention may be in these various lipid forms and is not limited to free fatty acids. Different types of lipid components can be extracted according to the extraction technique used. Lipids can be extracted with an effective amount of solvent. Suitable solvents may be determined by those skilled in the art. Polar lipids (eg, phospholipids) are generally extracted with polar solvents (eg, chloroform / methanol) and neutral lipids (eg, triglycerol) can generally be extracted with a non-polar solvent (eg, hexane). The preferred solvent may be pure hexane. A suitable ratio of hexane to dry biomass may be about 4 liters of hexane per kilogram of dry biomass. Hexane can be mixed with the biomass in a stirred reaction vessel, preferably at a temperature of about 50 DEG C for about 2 hours. After mixing, the biomass is filtered and separated from the oil containing hexane, and the hexane can be removed from the oil by a distillation technique known to those skilled in the art. Conventional oil seed processing equipment is suitable for performing filtration, separation and distillation. Additional processing steps known to those skilled in the art may be performed if necessary or appropriate for the particular application. Other methods of lipid recovery are described in the following references, which are incorporated herein by reference: PCT Publication No. WO 0176715 entitled " Method of fractionating oil and polar lipid-containing pure raw materials ", "Alcohol and centrifugation PCT Publication No. WO 01/076385 entitled " Process for fractionating oil and lipid-containing pure raw material ", PCT Publication No. WO 01/053512 entitled "

The present invention relates to a method for producing docosahexaenoic acid from microalgae, in which cells and lipids are simultaneously increased by converting the medium into a low salt and low nitrogen condition through a temporary supply of a medium containing only carbon source and no additional supply of nitrogen source and salt , It is possible to produce docosahexaenoic acid which is convenient and economical by improving the existing feed-in-oil culture method in which medium is continuously supplied.

Fig. 1 shows the result of measuring the concentration of microbial cells after the main cultivation in Example 1 and Comparative Example 1. Fig.
Fig. 2 shows DHA contents recovered from the cells after culturing in Example 1 and Comparative Example 1. Fig.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  One

The microalgae Aurantiochytrium limacinum SR21 (ATCC MYA-1381) was cultured. The preculture was incubated for 2 days in a 10 mL volumetric flask in a 50 mL Erlenmeyer flask at a temperature of 25 DEG C and a stirring speed of 200 rpm, and then cultured in a 250 mL Erlenmeyer flask in a volume of 50 mL for 2 days with shaking.

The cultivation was carried out in a 1 L fermenter in a volume of 300 mL. The culture medium was composed of glucose (60 g / L), CSL (20 g / L), monosodium glutamate (20 g / L) and sea salt (20 g / L) 5, the culture temperature was 25 ° C, the stirring speed was 250 rpm, and the aeration amount was 0.5 vvm.

After 48 hours of incubation, the same volume (300 mL) of carbon source medium as the culture volume was temporarily added. The carbon source medium supplied was composed of glucose (58 g / L).

Comparative Example  One

The cells were cultured under the same conditions as in Example 1 at a volume of 2 × (600 mL). The cells were continuously cultured under the initial conditions without additional feeding medium. The final culture volume of Example 1 and Comparative Example 1 was the same at 600 mL.

Experimental Example  1. Microalgae cell concentration and DHA  Content check

1.1 Identification of microalgae cell concentration

After the main culture of Example 1 and Comparative Example 1, the culture broth was centrifuged to remove supernatant, and the cells were recovered, dried and weighed to measure the cell concentration (Fig. 1).

As a result, although the cell concentration was diluted to 1/2 with the addition of the carbon source medium (at the time of 48 hours of culturing), it was steadily increased thereafter to reach 22.6 g / L at the final cell concentration (Final cell concentration of Comparative Example 1 was 18.9 g / L).

1.2 Microalgae DHA  Content check

The fatty acid-containing retention test method, which is the standard of the Food and Drug Administration, was used for methyl esterification by using 0.5N sodium hydrogensulfate and 14% trifluoroborane methanol to obtain the cells from the cells cultured in Example 1 and Comparative Example 1 DHA was recovered and a fatty acid methyl esterified sample was analyzed by gas chromatography (use model: Agilent 7890A, use column: DB-23), and the peak area of DHA was compared with the peak area of the internal standard To determine the DHA content (FIG. 2 and Table 1).

division Cell density (g / L) Per cell DHA  content (%) DHA  Concentration (g / L) Yield (%) Example 1 22.6 24.9 5.63 38.3 Comparative Example 1 18.9 15.4 2.91 31.5

As a result, it was confirmed that the final DHA content of Example 1 was much higher than that of Comparative Example 1, and that it was improved by about 61.8%. In comparison with Comparative Example 1, the amount of nitrogen source and salt was 1/2, but the concentration of DHA production was increased about twice, and it was confirmed that the method of temporarily supplementing the carbon source was very effective for DHA accumulation.

Claims (14)

(a) culturing aurantiochytrium limacinum in a medium containing a carbon source and a nitrogen source at a temperature of 15 ° C to 34 ° C and a pH of 4 to 6 for 46 to 52 hours, and
(b) A composition containing a nitrogen source and a carbon source at a concentration of 50 to 60 g / L is temporarily supplied to the culture medium at 100 to 150% (v / v) of the medium volume, ≪ / RTI > to 69 for < RTI ID = 0.0 >
A method for producing docosahexaenoic acid (DHA) from Oran Shokitrium limassinum.
delete delete delete delete delete delete The method according to claim 1, wherein the carbon source is at least one selected from the group consisting of molasses, malt extract, starch, glucose, dextrin, sulfurous acid waste solution, fibrin, whey, alkaline, alcohol, fat and oil.
delete 4. The method of claim 1, wherein the orcansium limmiminum is pre-cultured in a medium comprising glucose, yeast extract and sea salt.
The method of claim 1, wherein the medium is a medium comprising glucose, CSL (corn steep solid), Monosodium glutamate and sea salt.
delete delete 3. The method of claim 1, further comprising recovering DHA from the culture in which Oranxactium Limassinum is cultured.

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