KR20140070181A - Method of producing microalgal dha - Google Patents

Abstract

The present invention relates to a method for producing docosahexaenoic acid (DHA) using microalgae and, more specifically, to a method for producing DHA using microalgae, including a primary culture of the microalgae in a high concentration of salt and a secondary culture of the microalgae in a low concentration of salt. The method can lead to increases in both microalgae cell growth and DHA productivity, prevent the corrosion of a reactor by applying a low-salt medium in a main culturing step corresponding to a direct culturing process, and reduce in the medium cost, thereby producing economical microalgae DHA.

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) using microalgae, and more particularly, to a method for producing microcapsules containing microalgae by primary culturing at a high salt concentration and secondary culturing at a low salt concentration To a method for producing DHA using microalgae.

Omega-3 unsaturated fatty acids are also referred to as omega-3 highly unsaturated fatty acids and are typically represented by docosahexaenoic acid (DHA) Eicosapentaenoic acid (EPA), arachidonic acid (ARA), docosapentaenoic acid (DPA), and? -Linolenic acid are known. 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. 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. 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 in the genus Thraustochytrium and Schizochytrium, a kind of marine microalgae, is described in Ellenbogen et al. BB, S. Aaronson, S. Goldstein and M. Belsky, Comparative Biochemistry and Physiology, 29, 805-811 (1969).

In addition, Martek is a microorganism belonging to the genus Schizochytrium sp. ATCC 20888 (Schizochytrium sp. ATCC 20888) and Shizokatori sp. Discloses a method for preparing omega-3 unsaturated fatty acids using ATCC 20889 (Schizochytrium sp. ATCC 20889) (U.S. Patent No. 5,130,242 and U.S. Patent No. 5,340,742). In addition, Suntory has reported Schizochytrium limacinum SR21 (Japanese Patent Publication No. 1997-000284, U.S. Patent No. 6,582,941) as a microorganism having excellent productivity of docosahexaenoic acid, . It has been known that DHA content is highest when Schizochytrium limacinum SR21 is cultured with 2% seawater treatment (HJ Chin et al., Australian Journal of Agricultural Research, 2006, 57, 13-20).

A method for producing DHA using microalgae is disclosed in Korean Patent Publication No. 2007-0042115, which is a method of culturing microorganisms of the Thraustochytriaceae using an optimized low-salt medium. However, There was no increase in the productivity of the comparative cells. In addition, although the production of a high concentration of diethiene in microalgae using a deformation amount of chlorine and potassium is known (Korea Publication No. 2006-0097009), the control of specific salt concentration (chlorine, potassium) Showed the necessary problems.

The present invention relates to a DHA production method and a DHA production method using a salt concentration-controlled two-step cultivation method in order to increase microbial cell growth and DHA productivity, to avoid corrosion of a reactor by applying a low salt medium in a culture step, ≪ / RTI > produced by the method of the present invention.

The present invention has been made in view of the above problems, and it is an object of the present invention to provide a microalgae growth and DHA productivity In order to avoid the corrosion of the reactor by applying the low salt medium in the culture step and to reduce the cost of the medium and to improve the economical efficiency, a method of producing DHA by a two-stage culture method in which the salt concentration is adjusted and DHA produced by the above- .

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The present invention relates to a method for pre-culturing microalgae at a salt concentration of 27 g / L or more; And a second step of culturing the microalgae at a salt concentration of not more than 20 g / L, wherein the salt is preferably a salt of water, sodium chloride (NaCl) 77.7 to 79.5% by weight, of magnesium chloride as a salt (MgCl ₂₎ 10.8 to 10.9% by weight, and potassium sulfate (K ₂ SO ₄₎ 2.0 to 2.6 weight%, magnesium sulfate (MgSO ₄₎ 4.8 to 24.8% by weight, potassium chloride (KCl) 2.5 to 3.2 wt%, calcium chloride (CaCl ₂₎ 0.95 to 4.09 By weight of sodium hydrogen carbonate (NaHCO ₃ ), 0.6 to 3.2% by weight of sodium hydrogencarbonate (NaHCO ₃ ) and the balance salt.

By this method, microbial cell growth and DHA productivity can be increased at the same time. In this culture step corresponding to direct culture process, low-salt medium can be used to avoid the corrosion of the reactor and to produce economical microalgae DHA by lowering the medium cost .

Hereinafter, the present invention will be described in detail.

In the present invention, the microalgae cultivation step for DHA production is divided into two stages based on the salt concentration, the microalgae are cultured by applying the high salt concentration in the pre-culture stage, and then the microalgae are cultured by applying the low salt concentration in the culture stage A method for producing DHA using microalgae including a method for culturing microalgae.

According to the present invention, in the culturing of microalgae, the microalgae are pre-cultured at a relatively low salt concentration, and then the microalgae are cultured at a relatively low salt concentration. Thus, in this culture, microalgae growth and DHA production .

When the salt concentration is less than 27 g / L, the growth of microalgae is insufficient, and when the salt concentration is more than 20 g / L in the second step (culture), the production of DHA is low. In one embodiment of the present invention, the microalgae are pre-cultured in a culture condition having a salt concentration of 27 g / L or more to improve the growth of microalgae, and culturing the microalgae at a salt concentration of 20 g / L or less. And a method for producing the same.

In order to maximize the growth of microalgae to improve the production of DHA, the preferred salt concentration in the pre-culturing step may be 30 g / L or more, more preferably the salt concentration may be 35 g / L or more, It can be more than 37 g / L. The salt concentration in the pre-culturing step may be 40 g / L or more, for example.

To improve the production of DHA and prevent corrosion of the reactor, the preferred salt concentration of the present culturing performed after the pre-incubation may be 0 to 10 g / L, more preferably 0 to 7 g / L, The preferred culture conditions may be an acid-free condition.

The salt is a salt including sodium chloride (NaCl), MgCl ₂ (magnesium chloride), K ₂ SO ₄ (potassium sulfate), MgSO ₄ (magnesium sulfate) and the like, For example, artificial sea salt from Sigma-Aldrich.

Salt water one example applicable to the present invention is sodium chloride (NaCl) 77.7 to 79.5% by weight of magnesium chloride (MgCl ₂₎ 10.8 to 10.9% by weight, and potassium sulfate (K ₂ SO ₄₎ 2.0 to 2.6 weight%, magnesium sulfate (MgSO ₄₎ 4.8 to 24.8% by weight, potassium chloride (KCl) 2.5 to 3.2 wt%, calcium chloride (CaCl ₂₎ 0.95 to 4.09 By weight of sodium hydrogen carbonate (NaHCO ₃ ), 0.6 to 3.2% by weight of sodium hydrogencarbonate (NaHCO ₃ ) and the balance salt. It may also be an artificial sea salt produced by taking into account the salt type and concentration of the sea water salt.

Except for the salt, other ingredients of the medium may be ingredients known to promote growth and production of DHA to commercially viable levels and may also be in the form of pharmaceutical compositions such as those described in U.S. Patent Nos. 5,130,242, 5,407,957, 5,397,591, 5,492,938, and U.S. Patent No. 5,711,983. More specifically, a carbon source such as glucose, various starches, molasses, and ground corn may be used. An affinity organic or inorganic nitrogen source is also 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. For example, the medium may include glucose at 60 to 100 g / L as a carbon source and 10 to 50 g / L of corn steep liquor as a nitrogen source.

The microalgae can be cultured at aeration of 0.5 vvm and agitation at 350 rpm.

Further, other optimum conditions of the culture method can be easily 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. Preferably the temperature is maintained at about 20 [deg.] C to about 28 [deg.] C.

The pH is maintained at 4 to 6, although not particularly limited thereto.

Another embodiment of the present invention may further include a step of recovering DHA after the second step culturing.

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 a polar solvent (eg, chloroform / methanol) and neutral lipids (eg, triglycerol) are generally extracted with a nonpolar solvent (eg, hexane). A preferred solvent is pure hexane. The proper ratio of hexane to dry biomass is about 4 liters of hexane per kilogram of dry biomass. The hexane is preferably mixed with the biomass in a stirred reaction vessel at a temperature of about 50 DEG C for about 2 hours. After mixing, the biomass is filtered and separated from the hexane containing the oil. Hexane is 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 microalgae typically include all microalgae containing DHA and are preferably selected from the group consisting of Schizochytrium genus, Thraustochytriales genus, Ulkenia genus, Thraustochytrium ), Genus Japonochytrium , Crypthecodinium genus, Haliphthoros genus, Chlamydomonas genus, Aurantiochytrium genus and porphyridium genus Porphyridium ). ≪ / RTI > Preferably, the micro-algae in order to maximize the production of DHA may lie alveolar kit Leeum (Schizochytrium), most preferably Schizochytrium limacinum SR21.

When applying the method of producing DHA using microalgae according to an example of the present invention, the microalgae can produce DHA of 10% or more per dry weight of biomass.

The method of culturing microalgae according to the present invention increases the microalgae growth and DHA productivity at the same time by adjusting the salt concentration in the medium to a low concentration, The low-salt medium can be used to avoid the corrosion of the reactor and lower the cost of the medium, thereby enabling production of economical microalgae DHA.

FIG. 1 is a graph showing the effect of salt concentration of Schizochytrium limacinum SR21 cells.
FIG. 2 is a graph showing the effect of salt concentration of Schizochytrium limacinum SR21 cells.
FIG. 3 shows the DHA content according to the salt concentration of the pre-culture and the main culture.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

[Example]

Example  One

Schizochytrium limacinum SR21 strain (ATCC MYA-1381). The medium consisted of glucose (30 g / L), yeast extract (10 g / L), and salinity 0, 10, 20, 30 and 40 g / L of seawater salt (S9883). The strain was inoculated with the strains at a concentration of 6 to 7 g / L in the medium at 10 v / v%, and cultured at an initial pH of 5, a pre-culture temperature of 25 ° C and a stirring speed of 250 rpm. The cells were preincubated for 3 days, and after the preincubation, they were converted into the present cultures. After the pre-culture, the culture broth was centrifuged to remove the supernatant, and the cells were recovered, dried and weighed to measure the cell concentration. The results are shown in Fig. As shown in FIG. 1, the concentration of Schizochytrium limacinum SR21 tended to increase with increasing salt concentration.

The pre-cultured solution was inoculated at 10 v / v% for 5 days. Specifically, with respect to a culture carried out in sea water of a salt concentration 40 g / L of the pre-culture salt concentration, together with the pre-culture at different salt concentrations (0, 10, 20g / L ) Schizochytrium limacinum SR21 strain was cultured. The medium consisted of glucose (30 g / L), mono sodium glutamate (10 g / L) and seawater salt (0, 10 and 20 g / L) (sigma adhrich sea salts pH 5, the incubation temperature was 25 캜, and the agitation speed was 250 rpm.

Comparative Example  One

Except that the salt concentration of the pre-culture was 0, 10, 20 g / L, and the salt concentration of the culture was 0, 10, 20, 30 and 40 g / L, Lt; / RTI >

Experimental Example  1. Microalgae cell concentration and DHA  Content check

1.1 Identification of microalgae cell concentration

After the culturing 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. The results are shown in FIG. 2, Table 1 and Table 2 Respectively.

division Pre-culture
Salt concentration (g / L) Cultivation
Salt concentration (g / L) Cell density (g / L) DHA content (%) Example 1 40 0 5.70 ± 1.11 16.97 ± 3.27 10 5.33 ± 1.03 11.38 + - 0.61 20 3.90 + - 0.62 10.94 + - 0.73

division Pre-culture
Salt concentration (g / L) Cultivation
Salt concentration (g / L) Cell density (g / L) DHA content (%) Comparative Example 1 0 0 0.53 + - 0.45 6.80 ± 2.88 10 0.33 + - 0.25 6.78 ± 3.30 20 0.30 ± 0.17 6.59 ± 3.37 30 0.97 ± 0.59 4.77 ± 0.88 40 0.57 0.21 4.60 ± 0.88 10 0 0.97 + - 0.25 7.61 + - 2.37 10 0.67 ± 0.06 6.53 ± 2.08 20 0.73 ± 0.29 5.62 ± 0.68 30 0.97 + 0.15 5.33 ± 0.96 40 0.70 + - 0.10 4.65 ± 0.02 20 0 0.73 ± 0.15 6.60 ± 1.69 10 1.33 ± 1.02 7.43 ± 1.41 20 0.77 + - 0.21 5.17 ± 0.82 30 0.67 ± 0.12 5.22 ± 0.42 40 0.87 0.25 4.09 ± 0.10

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 to methylate with 0.5N methanolic sodium hydrogensulfate and 14% trifluoroborane methanol to obtain DHA from the cells cultured in Example 1 and Comparative Example 1 And the fatty acid methyl esterified sample was analyzed by gas chromatography (use model: Agilent 7890A, use column: DB-23), and the peak area of the internal standard substance measured through this was compared with the peak area of DHA (The mass of DHA was quantified by comparing the peak area of the internal standard material with the peak area of DHA during GC analysis), and the DHA content was calculated. The DHA content was calculated as shown in Equation 1 below, Are shown in Fig. 3, Table 1 and Table 2.

[Equation 1]

DHA content = < 100 X extracted DHA mass / dry cell mass used for extraction >

As shown in FIG. 2, Table 1 and Table 2, the pre-culture condition of 40 g / L of salt concentration had the greatest effect on the increase of Schizochytrium limacinum SR21 cell mass , Recorded Schizochytrium limacinum SR21 cell concentration and DHA production were increased. In particular, as shown in FIG. 3, Table 1 and Table 2, the DHA content was found to be about 17% at a salt concentration of 40 g / L and 0 g / L of the present culture, .

In addition, in Comparative Example 1 in which the salt concentration of the pre-culture was 0, 10, and 20 g / L, not only the cell concentration was low, but also the salt concentration of the present culture at 0, 10, 20, 30 and 40 g / It was confirmed that DHA production was lower than that of Example 1.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (9)

A first step of pre-culturing the microalgae under a salt concentration of 27 g / L or more; And a second step of culturing the microalgae at a salt concentration of 20 g / L or less. The present invention also provides a method for producing docosahexaenoic acid (DHA) using microalgae. The method of claim 1, wherein the salt is sodium chloride (NaCl) 77.7 to 79.5% by weight of magnesium chloride (MgCl ₂₎ 10.8 to 10.9% by weight, and potassium sulfate (K ₂ SO ₄₎ 2.0 to 2.6 weight%, magnesium sulfate (MgSO ₄₎ 4.8 to 24.8% by weight, potassium chloride (KCl) 2.5 to 3.2 wt%, calcium chloride (CaCl ₂₎ 0.95 to 4.09 % by weight of sodium hydrogen carbonate (NaHCO ₃₎ 0.6 to sea salt production method comprising a salt of 3.2% by weight and the remaining amount. The production method according to claim 1, wherein the salt concentration of the first step is 30 g / L or more. The production method according to claim 3, wherein the salt concentration of the first step is 35 g / L or more. The production method according to claim 1, wherein the salt concentration in the second step is 0 to 10 g / L. The production method according to claim 1, further comprising the step of recovering DHA from the culture obtained after the second step. The microalgae of claim 1, wherein the microalgae are selected from the group consisting of Schizochytrium sp., Thraustochytriales sp., Ulkenia sp., Thraustochytrium sp., Japonochytrium sp. A production selected from the group consisting of Crypthecodinium genus, Haliphthoros genus, Chlamydomonas genus, Aurantiochytrium genus and Porphyridium genus Way. The production method according to claim 1, wherein the production amount of the DHA is 10% by weight or more per dry weight of the biomass of the microalgae. The production method according to claim 1, wherein the microalgae is cultivated in a medium containing a carbon source and a nitrogen source.

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